Heme sequesting peptides and uses therefor

ABSTRACT

The disclosure relates to engineered heme sequestering peptides and their use in treating cancer and inhibiting microbial infections and colonization.

PRIORITY CLAIM

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 62/821,823, filed Mar. 21, 2019, the entirecontents of which are hereby incorporated by reference.

FUNDING

The subject matter of the present was developed with funding from theCancer Prevention and Research Institute of Texas under Grant No.RP160617.

BACKGROUND

Iron is an essential nutrient for virtually all living organisms due toits ability to accept and donate electrons with relative ease. In humansand mammals, 95% of functional iron is in the form of heme, particularlyhemoglobin, which account for two thirds of total body iron (Beutler etal., 2009). In the human blood, cell-free heme exists in micromolarconcentrations predominantly in the form of hemoglobin (Oh et al.,2016). Pathogenic microbes have developed sophisticated mechanisms toextract and use heme directly or as an iron source. One effectivemechanism of heme acquisition in pathogenic bacteria is the use ofhemophores, which are functionally analogous to siderophores (Wandersman& Delepelaire, 2012). Particularly, the HasA-type hemophores have beenidentified in many Gram-negative bacteria and bind to heme with veryhigh affinity (Kd=18 pM) (Deniau et al., 2003). HasA binds to hemoglobinand extracts heme and delivers heme to the bacterial outer membranereceptor HasR for internalization. In the same vein, pathogenic fungihave also developed sophisticated mechanisms to scavenge and extractheme from human hosts (Bairwa et al., 2017; Roy and Kornitzer, 2019).For example, Candida albicans expresses a network of heme-bindingproteins to acquire heme from hemoglobin (Kuznets et al., 2014).

In humans, it is increasingly clear that heme import and export arecritical for the proper functioning of many cells and tissues, includingerythroid and neuronal cells and systems (Khan and Quigley, 2011;Chiabrando et al., 2018; Reddi and Hamza, 2016). While most normal cellsin the human body are not exposed to blood directly, tumor cells can beexposed to blood and heme due to leaky vessels. Interestingly, recentstudies in the authors' lab showed that non-small cell lung cancer(NSCLC) cells exhibit intensified heme uptake relative to normal cells,leading to elevated and mitochondrial heme levels, mitochondrialoxidative phosphorylation, and ATP generation (Sohoni et al., 2019).Elevated ATP levels promote tumorigenic functions of NSCLC cells.Overexpression of heme uptake proteins further potentiate tumorigenicfunctions of NSCLC cells. Notably, lung cancer is the leading cause ofcancer-related death in the US. About 85-90% of cases are classified asNSCLC (Siegel et al., 2012). Despite the advent of targeted therapiesand immunotherapies, an effective treatment or cure for lung cancerremains an unlikely outcome for most patients. The five-year survivalrate remains 10-20%, lower than many other cancers, including breast(90%) and prostate (99%) cancers (American Cancer Society, 2019). Thus,novel therapeutic strategies are necessary for dramatically improvingthe survival rate of lung cancer patients. The elevated need of NSCLCcells for heme affords a new potential strategy for lung cancertreatment.

Heme is a central metabolic and signaling molecule that regulatesdiverse processes ranging from transcription to microRNA processing(Barr et al., 2012; Mense and Zhang, 2019; Chen and Zhang, 2019;Wissbrock et al., 2019; Small et al., 2009; Shimizu et al., 2019). Hemealso serves as a prosthetic group in proteins and enzymes involved inoxygen utilization and metabolism. Heme function and mitochondrialrespiration are tightly linked. Multiple subunits in oxidativephosphorylation (OXPHOS) complexes II-IV contain heme. Heme alsocoordinates the expression and assembly of OXPHOS complexes (Ortiz deMontellano, 2009; Kim et al., 2012; Padmanaban et al., 1989). Clearly,heme possesses unique signaling and structural properties that enable itto coordinate elevated OXPHOS in not only NSCLC cells, but also an arrayof drug-resistant cancer cells. Recent studies have shown thatdrug-resistant cells of acute and chronic myeloid leukemia, breastcancer, and melanoma depend on OXPHOS and that targeting oxidativemetabolism and mitochondrial respiration overcomes their drug resistance(Farge et al., 2017; Kuntz et al., 2017; Navarro et al., 2016; Zhang etal., 2016; Lee et al., 2017). Thus, it is likely that thesedrug-resistant cells depend on ample heme supply for their resilienttumorigenicity. Further, a plethora of epidemiological studies haveshown that elevated heme intake is associated with an array of commondiseases, including several types of cancer, diabetes, and heart disease(Hooda et al., 2014).

SUMMARY

In accordance with the present disclosure, there is provided arecombinant heme sequestering peptide (HeSP) comprising one or more of(a) one or more neutral amino acid substitutions in a heme bindingpocket; (b) a fusion of heme binding protein (HBP) sequences fromdistinct heme binding proteins; and/or (c) a heme binding protein (HBP)having a truncation and/or internal deletion that reduces theimmunogenicity of said heme binding protein. The HBP may compriseYersinia pestis HasA sequences. The HeSP may have a single neutral aminoacid substitution in a heme binding pocket, such as Q32H, or may haveonly two neutral amino acid substitutions, such as Q32H and Y75M, Q32Hand Y75H, or Q32H and Y75C. The HeSP may comprise distinct heme bindingprotein are selected from two or more of Yersinia pestis HasA, Erwiniacarotovora HasA, Pectobacterium carotvorum HasA and Pseudomonasfluorescens HasA. The HeSP may comprise a truncation that reducesimmunogenicity is a C-terminal truncation. The HeSP may comprise adeletion that reduces immunogenicity is in the C-terminal half of saidheme binding protein. The HeSP may exhibit (a) and (b), (b) and (c), (a)and (c), or all of (a), (b) and (c). The HeSP has the amino acidsequence of SEQ ID NOS: 2-10. The HeSP may be bound to zincprotopoprhyrin.

In another embodiment, there is provided a method of sequestering hemefrom an environment comprising contact said environment with an HeSP asdescribed herein. The environment may be a biological sample, a cellculture, a surface, such as a plate, tube or well surface, the surfaceof a medical device. The HeSP may be fixed to a support, such as acolumn matrix, a well, a plate, a slide, a tube, a dipstick, a bead, ora nanoparticle. The method may further comprise detecting sequesteredheme. The method may further comprise quantifying the detectedsequestered heme. The environment may be an air handling device orsystem, a heating/cooling device or system, a water processing device orsystem, a water storage device or system, a water transport device orsystem, or a food processing device or system.

In yet another embodiment, there is provided a method of treating adisease, disorder or condition comprising administering to said subjectan HeSP as described herein. The disease may be cancer, such as lungcancer (such as non-small cell lung cancer), colon cancer, head & neckcancer, brain cancer, liver cancer, pancreatic cancer, prostate cancer,ovarian cancer, testicular cancer, uterine cancer, breast cancer (suchas triple negative breast cancer), skin cancer (such as melanoma),lymphoma, or leukemia. The cancer may be a recurrent cancer, drugresistant cancer, primary cancer or metastatic cancer. The method mayfurther comprise treating said subject with another cancer therapy suchas chemotherapy, radiotherapy, immunotherapy, toxin therapy, hormonaltherapy, or surgery. The HeSP may be administered local to a cancersite, regional to a cancer site, or systemically.

The disease may be an infectious disease, such as a fungal disease. Themethod may further comprise treating said subject with anotheranti-fungal therapy. The HeSP may be administered local to a site ofinfection, regional to a site of infection, or systemically.

In a further embodiment, there is provided a method of diagnosing aheme/iron/lead-related disease or disorder in a subject or a samplecomprising contacting said sample or subject with an HeSP as describedherein.

One of ordinary skill in the art will appreciate that startingmaterials, biological materials, reagents, synthetic methods,purification methods, analytical methods, assay methods, and biologicalmethods other than those specifically exemplified can be employed in thepractice of the disclosure without resort to undue experimentation. Allart-known functional equivalents, of any such materials and methods areintended to be included in this disclosure.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of thedisclosure claimed.

Thus, it should be understood that although the present disclosure hasbeen specifically disclosed by particular embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis disclosure as defined by the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIGS. 1A-F. (FIG. 1A) The levels of heme biosynthesis are elevated,though to a varying degree, in NSCLC cell lines. (FIG. 1B) The levels ofheme uptake are elevated in NSCLC cell lines. (FIG. 1C) Elevated hemebiosynthesis and uptake can both contribute to increased hemeavailability to NSCLC cells. (FIG. 1D) The levels of heme degradationare elevated in certain NSCLC cell lines. (FIG. 1E) The levels oftransferrin receptor TFRC are not uniformly elevated in NSCLC celllines. M: molecular marker. (FIG. 1F) Consistent with elevated hemebiosynthesis and uptake, the levels of mitochondrial heme are elevatedin NSCLC cells. Data are plotted as mean±SD. For statistical analysis,the levels in tumorigenic cells were compared to the levels innon-tumorigenic cells with a Welch 2-sample t-test. *, p-value, 0.05,**, p-value <0.005.

FIGS. 2A-D. (FIG. 2A) Oxygen consumption rates are elevated, though to avarying degree, in NSCLC cell lines. (FIG. 2B) ATP levels are elevatedin NSCLC cell lines. (FIG. 2C) The levels of mitochondrial biogenesisregulator NRF1 are upregulated in NSCLC cell lines relative tonon-tumorigenic cell lines. (FIG. 2D) The levels of mitochondrialbiogenesis regulator TFAM are upregulated in NSCLC cell lines relativeto non-tumorigenic cell lines. M: molecular marker. Data are plotted asmean±SD. For statistical analysis, the levels in tumorigenic cells werecompared to the levels in non-tumorigenic cells with a Welch 2-samplet-test. *, p-value, 0.05, **, p-value <0.005.

FIGS. 3A-C. (FIG. 3A) Heme-sequestering peptides (HSPs) inhibit hemeuptake in NSCLC cell lines in a reversible manner. In the absence ofHSPs, the presence of 60 mM (1×ZnPP) or 120 mM (2×ZnPP) ZnPP resultedthe same levels of intracellular uptake, as uptake was saturated above60 mM. The inhibition of ZnPP uptake by HSPs occurred only at 1×ZnPP,whereas 2×ZnPP completely reversed the inhibition by HSP2, indicatingthat the presence of HSP2 did not cause irreversible effects on thecells. HSP contains HasA residues 1-193 with no mutations. (FIG. 3B)Mitochondrial heme levels in NSCLC cells gradually decrease as treatmentwith HSP2 continues. H1299 NSCLC cells bearing the expression plasmidfor the peroxidase reporter for mitochondrial heme were treated with 25mM HSP2 for the indicated time periods, and then reporter activitieswere measured as described in Methods. The activities were normalized toeGFP fluorescent signals which serve as a control for transfectionefficiencies. In FIG. 3B, values were presented as fold changes relativeto untreated cells. (FIG. 3C) HSP1 and HSP2 significantly reduceproliferation of NSCLC cell lines but not non-tumorigenic cell lineHBEC30KT (HBEC). Data are plotted as mean±SD. For statistical analysis,the levels in treated cells were compared to the levels in untreatedcells (0 hour) with a Welch 2-sample t-test. *, p-value, 0.05, **,p-value <0.005.

FIGS. 4A-G. HSPs inhibit migration capabilities (FIG. 4A) and invasioncapabilities (FIG. 4B) in H1299 NSCLC cells. Succinyl acetone (SA) isshown for comparison. Addition of 10 mM heme largely reversed theeffects of SA and HSPs. The images shown are cells that had migratedacross Transwell inserts (in FIG. 4A) or had crossed invasion chamberscoated with Corning Matrigel matrix and also passed Transwell inserts(in FIG. 4B). Data are plotted as mean±SD. For statistical analysis, thelevels in treated cells were compared to the levels in untreated cellswith a Welch 2-sample t-test. *, p-value, 0.05, **, p-value <0.005. Forheme add-back experiments, the levels in cells treated with heme andHSP2 were compared to the levels in cells treated with only HSP2. [**],p-value <0.005. (FIG. 4C) The effect of SA on colony formation in H1299cells. (FIG. 4D) The effect of HSP1 on colony formation in H1299 cells.(FIG. 4E) The effect of HSP2 on colony formation in H1299 cells. (FIG.4F) The effects of heme oxygenase inhibitor SnPP and ZnPP on colonyformation in H1299 cells. (FIG. 4G) The effect of DFX on colonyformation in H1299 cells.

FIGS. 5A-H. The effect of HSP2 on the growth and progression of H1299NSCLC lung tumor xenografts and on the levels of OXPHOS subunits intumors. (FIG. 5A) Representative bioluminescence images of mice bearingH1299 lung tumor xenografts treated without (Control) or with HSP2 (25mg/kg) or HSP2 (10 mg/kg) (n=6/group). Treatment started at 4.5 weeksafter tumor cell implantation when authentic BLI signals (>5×10⁶photons/second) were detected from tumors of all tested mice. Treatmentswere stopped, and mice were sacrificed after the untreated mice withtumors appeared moribund. (FIG. 5B) The quantified luminescence signalsrepresenting tumor volumes. Data are plotted as mean±SD. For statisticalanalysis, the levels in treated tumors were compared to the levels inuntreated tumors with a Welch 2-sample t-test. *, p-value <0.05. (FIG.5C) The body masses of mice under each treatment condition. (FIG. 5D)Representative H&E images of control tumors and tumors treated with HSP2or control saline. Tumors are marked with light blue outlines. Montage(scale bar: 2 mm), 10× (scale bar: 200 mm), and 40× (scale bar: 50 mm)images of the H&E sections are shown from left to right. The rectanglesin Montage and 10× denote the regions shown in 10× and 40×,respectively. (FIG. 5E) Representative IHC images of H1299 NSCLC tumortissue sections and graph showing the levels of COX5A in control andHSP2-treated tumors. (FIG. 5F) Representative IHC images of H1299 NSCLCtumor tissue sections and graph showing the levels of COX4I1 in controland HSP2-treated tumors. (FIG. 5G) Representative IHC images of H1299NSCLC tumor tissue sections and graph showing the levels of UQCRC2 incontrol and HSP2-treated tumors. (FIG. 5H) Representative IHC images ofH1299 NSCLC tumor tissue sections and graph showing the levels of CYCSin control and HSP2-treated tumors. Shown are montages and 10× images ofcontrol and HSP2-treated tumor tissue sections stained with DAPI orantibodies against the indicated protein. The light blue lines in DAPIimages outline the tumors in the lung. The white rectangles in DAPIimages denote the regions shown in 10× images. The heart was oftenstained and is marked with “H”. Scale bar: montage, 1 mm; 10×, 20 mm.Protein levels were quantified, and data are plotted as mean±SEM. Thevalues shown in the graphs are averages of signals quantified from threeindependent IHC experiments. For statistical analysis, the levels intreated tumors were compared to the levels in control tumors with aWelch 2-sample t-test. **, p-value <0.005.

FIGS. 6A-F. HSP2 effectively suppresses the growth, oxygen consumptionrates, and ATP generation in subcutaneous NSCLC tumor xenografts. (FIG.6A) Images showing dissected tumors from mice implanted withsubcutaneous xenografts (H1299-luc cells), treated with saline (control)or HSP2. Treatment started at 1.5 weeks after tumor implantation whenauthentic BLI signals were detected, and tumors were visible. Mice weresacrificed when tumors in untreated mice reach about 1 cm³. (FIG. 6B)The quantified bioluminescence signals representing tumor volumes frommice bearing subcutaneous xenografts treated with saline (control) orHSP2 (25 mg/kg I.V. every 3 days) (n=4/group). (FIG. 6C) Average massesfor control and HSP2-treated tumors (n=4/group). (FIG. 6D) Average bodymasses of control and HSP2-treated mice. (FIG. 6E) Oxygen consumptionrates (OCRs) measured in cells from control and HSP2-treated tumors.(FIG. 6F) ATP levels measured in cells from control and HSP2-treatedtumors. Data are plotted as mean±SEM. For statistical analysis, thelevels in HSP2-treated tumors were compared to the levels in controltumors with a Welch 2-sample t-test. **, p-value <0.005.

FIGS. 7A-H. The effects of overexpressing ALAS1 or SLC48A1 on hemesynthesis, uptake, migration, invasion, and tumor growth of NSCLC cells.(FIG. 7A) NSCLC cells overexpressing ALAS1 exhibit increased hemesynthesis. (FIG. 7B) NSCLC cells overexpressing SLC48A1 exhibitincreased heme uptake. (FIG. 7C) Migration is enhanced in NSCLC cellswith elevated heme synthesis or uptake due to overexpression of ALAS1 orSLC48A1. (FIG. 7D) Invasion is enhanced in NSCLC cells with elevatedheme synthesis or uptake due to overexpression of ALAS1 or SLC48A1.(FIG. 7E) Images showing tumor xenografts isolated from NOD/SCID miceimplanted with control NSCLC cells or cells overexpressing ALAS1 orSLC48A1. (FIG. 7F) Average masses of control tumors and tumors formed bycells overexpressing ALAS1 or SLC48A1 (n=5/group). (FIG. 7G) Oxygenconsumption rates (OCRs) measured in cells from control tumors and thosewith cells overexpressing ALAS1 or SLC48A1. (FIG. 7H) ATP levelsmeasured in cells from control tumors and those with cellsoverexpressing ALAS1 or SLC48A1. Data are plotted as mean±SEM. Forstatistical analysis, the levels in tumors formed by cellsoverexpressing ALAS1 or SLC48A1 were compared to the levels in controltumors with a Welch 2-sample t-test. **, p-value <0.005.

FIGS. 8A-B. The sequences of bacterial HasA proteins andheme-sequestering proteins (HeSPs). (FIG. 8A) The sequences of hemophoreHasA proteins from Yersinia pestis (SEQ ID NO: 1) and non-humanpathogens Erwinia carotovora (SEQ ID NO: 12), Pectobacterium carotovorum(SEQ ID NO: 13), and Pseudomonas fluorescens (SEQ ID NO: 14). Thesequences are divided into three segments. Key heme-binding residues 32(H or Q) and 75 (Y) are designated with red dots. Residues of theheme-binding pocket in HasA protein from Yersinia pestis are in red,conserved residues across the alignment are in green and conservativesubstitutions in blue. Residues are numbered according to the Yersiniapestis sequence. (FIG. 8B) Protein sequence maps of HeSPs generatedbased on HasA proteins from Yersinia pestis and non-pathogenic bacteria.

FIGS. 9A-C. HeSP2, like HasA_(yp), binds to heme strongly. (FIG. 9A) Thepull-down of HasA_(yp) and HeSP2 by heme agarose beads. Five hundredpicomoles of human serum albumin (HAS, positive control), carbonicanhydrase (CA, negative control), HasA_(yp), and HeSP2 were incubatedwith heme-agarose beads, respectively. The input (In) proteins, bound(Bu) and unbound proteins (UnB) were analyzed by SDS-PAGE and shown.(FIG. 9B), Absorption spectra of heme in the presence and absence ofHasA_(yp) or HeSP2. Line 1: 5 μM heme, line 2: 5 μM heme+10 μM HasA_(yp)or HeSP2, line 3: 10 μM HasA_(yp) or HeSP2. (FIG. 9C) Elution profilesof heme-HeSP mixtures on Sephadex-G50 columns. line1: 0.25 mM heme+0.25mM HasA_(yp) or HeSP2 (protein absorption at 280 nm), line2: 0.25 mMheme+0.25 mM HasA_(yp) or HeSP2 (heme absorption at 400 nm), line3: 0.25mM (heme absorption at 400 nm).

FIGS. 10A-D. (FIGS. 10A-C) HasA_(yp) or HeSP2 exhibit differentialsensitivity to chymotrypsin. β-amylase (FIG. 10A, for control), HasAyp(FIG. 10B), and HeSP2 (FIG. 10C) were incubated with or without heme,then chymotrypsin was added to the proteins with increasingconcentrations. Untreated (lanes 1 and 7) and chymotrypsin-treatedproteins (lanes 2-6 and 8-12) were analyzed by SDS-PAGE. Chymotrypsinconcentrations: lanes 2 and 8, 6.25 μg/ml; lanes 3 and 9, 12.5 μg/ml;lanes 4 and 10, 25 μg/ml; lanes 5 and 11, 50 μg/ml; lanes 6 and 12, 100μg/ml. (FIG. 10D) Detection of heme transfer from hemoglobin (Hb) toHeSP2. Hemoglobin (Hb), Apo-HeSP2, and mixtures of Hb and Apo-HeSP2 wereanalyzed on native PAGE and stained for protein and heme, respectively.Lane 1, 20 μM Hb; lane 2, 200 μM HeSP2; lane 3, 200 μM HeSP2+200 μMHeme; lane 4, 20 μM Hb+20 μM HeSP2; lane 5, 20 μM Hb+50 μM HeSP2; lane6, 20 μM Hb+100 μM HeSP2; lane 7, 20 μM Hb+200 μM HeSP2.

FIGS. 11A-B. The effects of HeSPs on NSCLC cell proliferation andsurvival. (FIG. 11A) HeSPs inhibit NSCLC cell proliferation and additionof heme largely reverses the inhibition. NSCLC cells were treated with20 μM HeSPs in the presence or absence of 20 μM heme in medium. Data areplotted as mean±SD. For statistical analysis, the levels in treatedcells were compared to the levels in untreated cells with a Welchtwo-sample t-test. (**, P<0.005). (FIG. 11B) HeSP2 induces apoptosis inNSCLC cell line and addition of heme largely reverses this effect. TheNSCLC cell line H1299 were treated with 20 μM HeSP2 in the presence orabsence of 20 μM heme. Then cells were subjected to apoptosis assayusing Annexin V-FITC and Propidium Iodide (PI) staining. The images ofcells were captured with bright field microscopy (BF) or fluorescentmicroscopy with a GFP or Texas Red (for PI) filters. Scale bar, 100 μm.

FIGS. 12A-E. HeSP2 effectively suppresses the growth, oxygen consumptionrates (OCRs), and ATP generation in subcutaneous NSCLC tumor xenografts.(FIG. 12A) Images showing resected tumors from mice implanted withsubcutaneous xenografts (H1299-luc cells) treated with saline (control)or the indicated HeSP. Treatment started at 1.5 weeks after tumorimplantation when authentic BLI signals were detected, and tumors werevisible. Mice were sacrificed and tumors were resected when tumors inuntreated mice reached about 1 cm³. (FIG. 12B) Average masses forcontrol and HeSPs-treated tumors (n=5/group). (FIG. 12C) Average bodymasses of control and HeSPs-treated mice. (FIG. 12D) OCR measured incells from control and HeSPs-treated tumors. (FIG. 12E) ATP levelsmeasured in cells from control and HeSP-treated tumors. Data are plottedas mean±SEM. For statistical analysis, the levels in HeSP-treated tumorswere compared with the levels in control tumors with a Welch two-samplet-test (*, P<0.05; **, P<0.005).

FIGS. 13A-B. The effects of HeSPs on C. albicans cell proliferation andbiofilm formation. (FIG. 13A) HeSPs inhibit C. albicans cellproliferation, and addition of heme largely reverses the inhibition. C.albicans cells were treated with 100 nM HeSPs in the presence or absenceof 100 nM heme. (FIG. 13B) HeSPs inhibit C. albicans biofilm formationand addition of heme largely reverses the biofilm formation. Biofilm wasformed in the presence or absence of 250 nM HeSPs with or without 250 nMheme. For statistical analysis, the levels in treated cells werecompared to the levels in untreated cells with a Welch two-samplet-test. (**, P<0.005).

FIG. 14. Liver function test results form HSP2-treated mice. The lungcancer model, as described in the Examples, was generated by orthotopicimplantation of H1299 tumor cells into NOD/SCID mice. The HSP2 peptidewas administered at 25 mg/kg every three days for four weeks.

FIGS. S1A-C. (FIG. S1A) The levels of the rate-limiting hemebiosynthetic enzyme ALAS1 are elevated in NSCLC cells. (FIG. S1B) Thelevels of heme transporter SLC46A1 are elevated in NSCLC cells. (FIG.S1C) The levels of heme degradation enzyme HMOX1 are elevated in NSCLCcells. Data are plotted as mean±SD. For statistical analysis, the levelsin tumorigenic cells were compared to the levels in non-tumorigeniccells with a Welch 2-sample t-test. *, p-value, 0.05, **, p-value<0.005.

FIGS. S2A-H. The levels of heme in subcellular organelles in NSCLC celllines. (FIG. S2A) Heme levels in cytoplasm. (FIG. S2B) Heme levels innucleus. (FIG. S2C) Heme levels in endoplasmic reticulum. (FIG. S2D)Heme levels in Golgi. (FIG. S2E) Heme levels in plasma membrane. (FIG.S2F) The levels of OXPHOS complex subunit cytochrome c (CYCS) areelevated in NSCLC cells. (FIG. S2G) The levels of OXPHOS complex subunitCOX4I1 are elevated in NSCLC cells. (FIG. S2H) The levels ofheme-containing enzyme cyclooxygenase (PTGS2) are elevated in NSCLCcells. Data are plotted as mean±SD. For statistical analysis, the levelsin tumorigenic cells were compared to the levels in non-tumorigeniccells with a Welch 2-sample t-test. *, p-value, 0.05, **, p-value<0.005.

FIGS. S3A-B. (FIG. S3A) NSCLC cell lines exhibit a varying degree ofmigration capabilities. (FIG. S3B) NSCLC cell lines exhibit a varyingdegree of invasion capabilities. The images shown are cells that hadmigrated across Transwell inserts (in FIG. S3A) or had crossed invasionchambers coated with Corning Matrigel matrix and passed Transwellinserts (in FIG. S3B). At least three independent experiments werecarried out for every condition. Data are plotted as mean±SD. Forstatistical analysis, the levels in NSCLC cells were compared to thelevels in HCC cells with a Welch 2-sample t-test.*, p-value, 0.05, **,p-value <0.005.

FIGS. S4A-E. Proteins and enzymes relating to heme and mitochondrialfunctions are upregulated in human NSCLC tissues relative to controlnormal tissues. The examined proteins and enzymes include therate-limiting heme synthetic enzyme ALAS1 (FIG. S4A), the hemetransporter SLC48A1 (FIG. S4B), the hemoprotein OXPHOS subunitcytochrome c (CYCS) (FIG. S4C), hemoprotein PTGS2 (FIG. S4D), and theregulator promoting mitochondrial biogenesis TFAM (FIG. S4E). Shown arethe representative Montages and 10× images of control and NSCLC tumortissue sections stained with DAPI or antibodies against the indicatedproteins. The yellow rectangles in Montages denote the regions shown in10× images. Scale bar: montage, 1 mm; 10×, 20 μm. Protein levels werequantified with cellSens dimension software (Olympus). The values shownin the graphs are averages of signals quantified from six control andNSCLC independent tissue slides, respectively. Signals were calculatedas described in Methods. Data are plotted as mean±SD. For statisticalanalysis, the levels in treated tumors were compared to the levels inuntreated tumors with a Welch 2-sample t-test. **, p-value <0.005.

FIGS. S5A-G. (FIG. S5A) The quantified luminescence signals representingtumor volumes from mice bearing orthotopic H1299 tumor xenograftstreated with saline (control), and SA (50 mg/kg, I.V.) every 3 days(n=6/group). Treatments started 4 days after cell implantation. Data areplotted as mean±SD. For statistical analysis, the levels in treatedtumors were compared to the levels in untreated tumors with a Welch2-sample t-test. The difference between control and SA was notstatistically significant. (FIG. S5B) The sequences of Y. pestis HasA(SEQ ID NO: 15) and HSPs. The changed amino acid residues are indicated.(FIG. S5C) HSP1 and HSP2 bind to heme beads. CA (carbonic anhydrase)serves as the negative control while BSA serves as the positive control.These were used and described previously by Lal et al., Nucleic AcidsRes, 46: 215-228 (2018). (FIG. S5D) HSP2 remains in the medium evenafter prolonged incubation with NSCLC cells. H1299 NSCLC cells wereincubated with 40 μM HSP2 in the medium for the indicated time periods.Then, the proteins in 5 μl medium was analyzed on SDS-PAGE gels. Mediumand HSP2 are usually refreshed after 3 days. Shown in the same gel aresamples from RPMI medium (RPMI), RPMI medium with HSP2, and RPMImedium+serum. (FIG. S5E) Quantified levels of HSP2 in the mediumrelative to the major serum protein albumin. For statistical analysis,the levels in 1-72 hours of incubation were compared to the levels in 0hour of incubation with a Welch 2-sample t-test. The variations are notstatistically significant. (FIG. S5F) The DIC and fluorescent images ofcells incubated with ZnPP or ZnPP+HSP2. H1299 NSCLC cells were incubatedwith 40 μM ZnPP or 40 μM ZnPP+HSP2 for 12 hours. Cells were washed toreduce background fluorescence from the medium before imaging. DIC andfluorescent (from ZnPP) images of the cells were taken and shown here.ZnPP bound with HSP2 did not enter the cells. (FIG. S5G) ZnPP in theabsence of HSP2 entered NSCLC cells and co-localized with mitotrackergreen.

FIGS. S6A-D. HSPs inhibit NSCLC cell proliferation in a dose-dependentmanner. (FIG. S6A) The same doses of HSP1 and HSP2 that affect NSCLCcell lines (FIGS. S6B-D) do not affect significantly the HBEC30KT celllines representing normal lung epithelial cells. Data fromrepresentative NSCLC cell lines HCC4017 (FIG. S6B), H1299 (FIG. S6C),and A549 (FIG. S6D) are shown. For statistical analysis, the levels intreated cells were compared to the levels in untreated cells with aWelch 2-sample t-test. **, p-value <0.005.

FIGS. S7A-I. HSPs inhibit tumorigenic functions in A549 NSCLC cells.(FIG. S7A) HSPs inhibit migration in A549 NSCLC cells. (FIG. S7B) HSPsinhibit invasion in A549 NSCLC cells. The potent inhibitor of hemesynthesis, succinyl acetone (SA), is shown for comparison. Addition of10 μM heme largely reversed the effects of SA and HSPs. The images shownare cells that had migrated across Transwell inserts (in FIG. S7A) orhad crossed invasion chambers coated with Corning Matrigel matrix andalso passed Transwell inserts (in FIG. S7B). Data are plotted asmean±SD. For statistical analysis, the levels in treated cells werecompared to the levels in untreated cells with a Welch 2-samplet-test.*, p-value, 0.05, **, p-value <0.005. For heme add-backexperiments, the levels in cells treated with heme and SA or HSPs werecompared to the levels in cells treated with only SA or HSPs. [**],p-value <0.005. The effects SA, HSPs, heme oxygenase inhibitors, andiron chelator DFX on NSCLC cell colony formation are shown in FIGS.S7C-G. (FIG. S7C) The effect of SA on colony formation in A549 cells.(FIG. S7D) The effect of HSP1 on colony formation in A549 cells. (FIG.S7E) The effect of HSP2 on colony formation in A549 cells. (FIG. S7F)The effects of heme oxygenase inhibitor SnPP and ZnPP on colonyformation in A549 cells. (FIG. S7G) The effect of DFX on colonyformation in A549 cells. (FIG. S7H) Comparison of the effects of HSP2and DFX treatments on the levels of transferrin receptor TFRC. (FIG.S7I) Comparison of the effects of HSP2 and DFX treatments on the levelsof ferroportin SLC40A1. In H & I, data from Western blotting analysis ofproteins prepared from NSCLC cells treated with HSP2 or DFX for 6 dayswere shown. For statistical analysis, the levels in treated cells werecompared to the levels in untreated cells (Control) with a Welch2-sample t-test. **, p-value <0.005.

FIGS. S8A-J. The effect of HSP2 on the growth and progression of H1299NSCLC lung tumor xenografts and on blood and liver functions in mice.(FIG. S8A) The quantified luminescence signals representing tumorvolumes. Treatment started at week 3 when tumors have grownsignificantly and BLI signals were about 5×10⁷ photons/seconds. Data areplotted as mean±SD. For statistical analysis, the levels in treatedtumors were compared to the levels in untreated tumors with a Welch2-sample t-test. *, p-value <0.05. (FIG. S8B) Representative H&E imagesof control tumors and tumors treated with HSP2 or control saline. Tumorsare marked with light blue outlines. Scale bar: 2 mm. (FIG. S8C) Averagered blood cell count in mice bearing orthotopic lung tumor xenograftstreated with or without HSP2. (FIG. S8D) Average hemoglobin levels inmice bearing orthotopic lung tumor xenografts treated with or withoutHSP2. (FIG. S8E) Average serum ALT (alanine transaminase) levels in micebearing orthotopic lung tumor xenografts treated with or without HSP2.(FIG. S8F) Western blots showing the levels of ALAS1 in H1299 cellsbearing the control or overexpression vector for ALAS1. (FIG. S8G)Western blots showing the levels of SLC48A1 in H1299 cells bearing thecontrol or overexpression vector for ALAS1. (FIG. S8H) Oxygenconsumption rates are increased in cells overexpressing ALAS1 orSLC48A1. (FIG. S8I) overexpression of ALAS1 or SLC48A1 promotes colonyformation by H1299 NSCLC cells. For statistical analysis, the levels incells overexpressing ALAS1 or SLC48A1 were compared to the levels incontrol cells with a Welch 2-sample t-test. **, p-value <0.005. (FIG.S8J) The levels of heme synthesis in H1299 cells cultured in normalmedium and heme-depleted medium (H dep), respectively. The level of hemesynthesis in heme-depleted medium presumably represents the totalcellular heme levels needed for the cells. The data show that de novoheme synthesis accounts for approximately 68% of total cellular hemelevels in H1299 cells, indicating that heme uptake accounts for 32%.Data are plotted as mean±SD. For statistical analysis, the levels inheme-depleted medium were compared to the levels in normal medium with aWelch 2-sample t-test.*, p-value, 0.05, **, p-value <0.005.

FIGS. S9A-B. HasA_(yp) does not suppress NSCLC tumor growthsignificantly. (FIG. S9A) Images showing resected tumors from miceimplanted with subcutaneous xenografts (H1299-luc cells) treated withsaline (control) or HasA_(yp). (FIG. S9B) Average masses for control andHasA_(yp)-treated tumors (n=4/group).

FIGS. S10A-B. (FIG. S10A) The transfer of heme from hemoglobin (Hb) toHeSP2 is instantaneous. Hemoglobin (Hb), Apo-HeSP2, and mixtures of Hband Apo-HeSP2 were incubated for the indicated times and then analyzedon native PAGE, followed by staining for protein and heme, respectively.Lane 1, 20 μM Hb; lane 2, 200 μM HeSP2; lane 3, 200 μM HeSP2+200 μMHeme; lanes 4-9, 20 μM Hb+200 μM HeSP2 incubated for 1, 2, 5, 10, 20,and 30 minutes. (FIG. S10B) Detection of heme transfer from hemoglobin(Hb) to HasA_(yp). Hemoglobin (Hb), apo-HasA_(yp), and mixtures of Hband HasA_(yp) were analyzed on native PAGE and stained for protein andheme, respectively. Lane 1, 20 μM Hb; lane 2, 200 μM HasA_(yp); lane 3,200 μM HasA_(yp)+200 μM Heme; lane 4, 20 μM Hb+20 μM HasA_(yp); lane 5,20 μM Hb+50 μM HasA_(yp); lane 6, 20 μM Hb+100 μM HasA_(yp); lane 7, 20μM Hb+200 μM HasA_(yp).

FIG. S11. The pull-down of various HeSPs by heme agarose beads. Fivehundred picomoles of indicated HeSPs were incubated with heme-agarosebeads. The input (In) proteins, bound (Bu) and unbound proteins (UnB)were analyzed by SDS-PAGE and shown.

FIGS. S12A-F. The effects of HeSPs on heme absorption spectra: line1: 5μM heme, line2: 5 μM heme+10 μM HeSP, line3: 10 μM HeSP.

FIGS. S13A-F. Elution profiles of heme-HeSP mixtures on Sephadex-G50columns: line1: 0.25 mM heme+0.25 mM HeSP (protein absorption at 280nm), line2: 0.25 mM heme+0.25 mM HeSP (heme absorption at 400 nm),line3: 0.25 mM (heme absorption at 400 nm).

FIGS. S14A-B. (FIG. S14A) The effects of HeSPs on heme uptake in H1299NSCLC cells. Cells were treated with 60 μmol/L ZnPP in the presence orthe absence of 40 μmol/L HeSPs. Data are plotted as mean±SD. Forstatistical analysis, the levels in treated cells were compared with thelevels in untreated cells with a Welch two-sample t-test (*, P<0.05; **,P<0.005). (FIG. S14B) HeSP2 remains stable in the medium duringprolonged incubation with NSCLC cells. H1299 NSCLC cells were incubatedwith 40 μM HeSP2 in the medium for the indicated time periods. Then, theproteins in 5 μl medium were analyzed on SDS-PAGE gels. Shown in thesame gel are samples from RPMI medium (RPMI), RPMI medium with HeSP2 andRPMI medium+serum. Quantified levels of HeSP2 in the medium relative tothe major serum protein albumin. For statistical analysis, the levels in1-48 hours of incubation were compared to the levels in 0 hour ofincubation with a Welch two-sample t-test. The variations are notstatistically significant.

FIGS. S15A-G. HeSPs inhibit NSCLC cell proliferation in a dose-dependentmanner. H1299 NSCLC cells were treated with various concentration of thefollowing HeSPs: (FIG. S15A) HeSP2, (FIG. S15B) HeSP2H, (FIG. S15C)HeSP2C, (FIG. S15D) HeSP2del, (FIG. S15E) HeSP2pc, (FIG. S15F) HeSP2ec,and (FIG. S15G) HeSP2pf. For statistical analysis, the levels in treatedcells were compared to the levels in untreated cells with a Welchtwo-sample t-test. (*, P<0.05; **, P<0.005).

FIG. S16. HeSPs induces apoptosis in H1299 NSCLC cells, and addition ofheme largely reverses the apoptosis. H1299 NSCLC cells were treated with20 μM HeSPs in the presence or absence of 20 μM heme. Cells weresubjected to apoptosis assay with Annexin V-FITC and Propidium Iodide(PI) staining. The images of cells were captured with bright fieldmicroscopy (BF) or fluorescent microscopy with a GFP or Texas Red (forPI) filters. Scale bar, 100 μm.

FIGS. S17A-D. HeSPs do not cause significant toxic effects on mouseblood and liver. (FIG. S17A) Average red blood cell counts in micebearing subcutaneous lung tumor xenografts treated with or withoutHeSPs. (FIG. S17B) Average hemoglobin levels in mice bearingsubcutaneous lung tumor xenografts treated with or without HeSPs. (FIG.S17C) Average serum ALT (alanine transaminase) levels in mice bearingsubcutaneous lung tumor xenografts treated with or without HeSPs. (FIG.S17D) Relative liver ATP levels in live cells from mice bearingsubcutaneous lung tumor xenografts treated with or without HeSPs.

FIG. S18. HeSPs inhibit heme uptake in Candida albicans. C. albicanscells were treated with 60 μmol/L ZnPP in the presence or the absence of40 μmol/L HeSPs. Data are plotted as mean±SD. For statistical analysis,the levels in treated cells were compared with the levels in untreatedcells with a Welch two-sample t-test (*, P<0.05; **, P<0.005).

DETAILED DESCRIPTION

The inventor hypothesized that lowering heme supply via hemesequestration is likely to be a useful strategy to manage and treat anarray of pathological conditions, including cancer. In this report, shedescribes studies to take advantage of bacterial hemophore HasA(Wandersman and Delepelaire, 2012; Huang and Wilks, 2017) to sequesterheme and make it unavailable for tumor cells and perhaps pathogenicfungi, because HasA delivers heme only to bacteria, not host cells.While wild-type HasA is efficient for scavenging heme and deliveringheme to bacteria, it may not have the best properties for hemesequestration. She reasoned that certain mutant HasA proteins thatretain high heme-binding affinity but having altered properties ininteraction with heme may be more effective in heme sequestration. Basedon the sequence of HasA proteins from Yersinia pestis and otherbacteria, a series of heme-sequestering proteins (HeSPs) were generatedand verified their interaction with heme. Further, it was found thatthese HeSPs can strongly suppress the growth of lung tumors in humanxenograft tumor models and inhibit the proliferation and biofilmformation in Candida albicans. These data show that heme sequestrationcan be an effective strategy for anti-cancer and anti-microbialtherapies. These and other aspects of the disclosure are set out below.

I. HEME BINDING PROTEINS

Free iron is limited in vertebrate hosts, thus an alternative tosiderophores has been developed by pathogenic bacteria to access hostiron bound in protein complexes. Heme binding protein A, or HasA, is asecreted hemophore that has the ability to obtain iron from hemoglobin.Once bound to HasA, the heme is shuttled to the receptor HasR, whichreleases the heme into the bacterium. A variety of differentmicroorganisms express a HasA molecule, including Yersinia pestis,Erwinia carotovora, Pectobacterium carotvorum and Pseudomonasfluorescens.

A. Engineered Heme Sequestering Peptides

The present inventor has designed heme sequestering peptides (HeSP's) toimprove their utility as therapeutic agents. For example, the inventorhas designed HeSP's using structural comparisons of known HasA's and acomputational algorithm based on coevolution to identify residues whosemutations may alter but not disrupt heme-binding properties, such as Y.pestis HasA Q32 and Y75, which may be substituted independently with Mor C. HeSPs with variations as these residues bind to heme strongly. Thealtered amino acids are known to coordinate heme well and thus thechanges are not expected to reduce heme binding. HeSPs with thesechanges have enhanced capabilities to inhibit heme uptake in NSCLCcells, in some cases by 5-fold.

Another concept involves engineering chimeric HasA molecules thatcontain HasA sequences from more than one organism. Examples shown belowemploy Yersinia pestis sequences in the amino-terminal ½ ro ¾ of themolecule, and sequences from Erwinia carotovora, Pectobacteriumcarotvorum and Pseudomonas fluorescens being used to replace the missingC-terminal Y. pestis sequences. This sort of modification can beemployed to reduce immunogenicity of the HeSP's. Similarly, the deletionof certain residues in HasA that may by hyper-immunogenic in mammaliansubjects is contemplated. Studies have identified putative epitopes inthe C-terminal half of Y. pestis HasA that are target for deletion.

B. HeSPs

The following sequences illustrate from native Y. pestis HasA anddesigned variants thereof. Underlined residues are substitutions ascompared to the native Y. pestis HasA sequence, bold residues representnon-Y. pestis sequences and lined-through residues are deleted ascompared to native Y. pestis HasA sequence.

HasA from Yersinia pestis (SEQ ID NO: 1)MSTTIQYNSNYADYSISSYLREWANNFGDIDQAPAETKDRGSFSGSSTLFSGTQYAIGSSHSNPEGMIAEGDLKYSFMPQHTFHGQIDTLQFGKDLATNAGGPSAGKHLEKIDITFNELDLSGEFDSGKSMTENHQGDMHKSVRGLMKGNPDPMLEVMKAKGINVDTAFKDLSIASQYPDSGYMSDAPMVDTVGVVDCHDMLLA A* HasAyp(SEQ ID NO: 2) MSTTIQYNSNYADYSISSYLREWANNFGDIDQAPAETKDRGSFSGSSTLFSGTQYAIGSSHSNPEGMIAEGDLKYSFMPQHTFHGQIDTLQFGKDLATNAGGPSAGKHLEKIDITFNELDLSGEFDSGKSMTENHQGDMHKSVRGLMKGNPDPMLEVMKAKGINVDTAFKDLSIASQYPDSGYMSDAPMVDTV* HeSP1 (SEQ ID NO: 3)MSTTIQYNSNYADYSISSYLREWANNFGDIDHAPAETKDRGSFSGSSTLFSGTQYAIGSSHSNPEGMIAEGDLKYSFMPQHTFHGQIDTLQFGKDLATNAGGPSAGKHLEKIDITFNELDLSGEFDSGKSMTENHQGDMHKSVRGLMKGNPDPMLEVMKAKGINVDTAFKDLSIASQYPDSGYMSDAPMVDTV*(Q in HasAyp is changed to H in HeSPs) HeSP2 (SEQ ID NO: 4)MSTTIQYNSNYADYSISSYLREWANNFGDIDHAPAETKDRGSFSGSSTLFSGTQYAIGSSHSNPEGMIAEGDLKMSFMPQHTFHGQIDTLQFGKDLATNAGGPSAGKHLEKIDITFNELDLSGEFDSGKSMTENHQGDMHKSVRGLMKGNPDPMLEVMKAKGINVDTAFKDLSIASQYPDSGYMSDAPMVDTV* HeSP2H (SEQ ID NO: 5)MSTTIQYNSNYADYSISSYLREWANNFGDIDHAPAETKDRGSFSGSSTLFSGTQYAIGSSHSNPEGMIAEGDLKHSFMPQHTFHGQIDTLQFGKDLATNAGGPSAGKHLEKIDITFNELDLSGEFDSGKSMTENHQGDMHKSVRGLMKGNPDPMLEVMKAKGINVDTAFKDLSIASQYPDSGYMSDAPMVDTV* HeSP2C (SEQ ID NO: 6)MSTTIQYNSNYADYSISSYLREWANNFGDIDHAPAETKDRGSFSGSSTLFSGTQYAIGSSHSNPEGMIAEGDLKCSFMPQHTFHGQIDTLQFGKDLATNAGGPSAGKHLEKIDITFNELDLSGEFDSGKSMTENHQGDMHKSVRGLMKGNPDPMLEVMKAKGINVDTAFKDLSIASQYPDSGYMSDAPMVDTV* HeSP2del (SEQ ID NO: 7)MSTTIQYNSNYADYSISSYLREWANNFGDIDHAPAETKDRGSFSGSSTLFSGTQYAIGSSHSNPEGMIAEGDLKMSFMPQHTFHGQIDTLQFGKDLATNAGG

MLEVMKAKGINVDTAFKDLSIASQYPDSGYMSDAPMVDTV* HeSP2ecFusion protein of Y. pestis HeSP2 1-128aa + wild-type Erwinia carotovora HasA 133-196aa (SEQ ID NO: 8)MSTTIQYNSNYADYSISSYLREWANNFGDIDHAPAETKDRGSFSGSSTLFSGTQYAIGSSHSNPEGMIAEGDLKMSFMPQHTFHGQIDTLQFGKDLATNAGGPSAGKHLEKIDITFNELDLSGEFDSGLTVSDRGVVHDVIYGLMGGQVQPLLDALTNAGIDINASLDSLSFATATSDAALSADTVVDVVGV* HeSP2pcFusion protein of Y. pestis HeSP2 1-136aa + wild-type Pectobacterium carotovorum HasA 139-196aa (SEQ ID NO: 9)MSTTIQYNSNYADYSISSYLREWANNFGDIDHAPAETKDRGSFSGSSTLFSGTQYAIGSSHSNPEGMIAEGDLKMSFMPQHTFHGQIDTLQFGKDLATNAGGPSAGKHLEKIDITFNELDLSGEFDSGKSMTENHQGVVHDVIYGLMSGQVQPLLDALTNAGIDINASLDSLSFATATSDAALSADTVVDVVGV* HeSP2pfFusion protein of Y. pestis HeSP2 1-101aa + wild-type Pectobacterium fluorenscens HasA 104-194aa (SEQ ID NO: 10)MSTTIQYNSNYADYSISSYLREWANNFGDIDHAPAETKDRGSFSGSSTLFSGTQYAIGSSHSNPEGMIAEGDLKMSFMPQHTFHGQIDTLQFGKDLATNAGSNYNLVSQEVSFTNLGLNSLKEEGRAGEVHKVVYGLMSGDSSALAGEIDALLKAIDPSLSVNSTFDDLAAAGVAHVNPAAAAAADVGLVGV*

C. Synthesis and Purification

Because of the size of the HeSP's, recombinant expression is thepreferred method for synthesis. A variety of commercially availablevectors and expression systems can be employed to generate the HeSP'sincluding those designed for mammalian cells, insect (Spodoptera;Baculovirus delivered) cells and bacterial cells. A preferred method isthe E. coli pET11a expression system. Baculovirus system may be used infuture to avoid endotoxin contamination in human trials.

In certain embodiments, the HeSP's are purified. The term “purified,” asused herein, is intended to refer to a composition, isolatable fromother components, wherein the protein is purified to any degree relativeto its naturally-obtainable state. A purified protein therefore alsorefers to a protein, free from the environment in which it may naturallyoccur. Where the term “substantially purified” is used, this designationwill refer to a composition in which the protein or peptide forms themajor component of the composition, such as constituting about 50%,about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about99% or more of the proteins in the composition.

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe cellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. Other methods for protein purification include,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; gel filtration, reversephase, hydroxylapatite and affinity chromatography; and combinations ofsuch and other techniques. In purifying a protein, it may be desirableto extract the protein using denaturing conditions. The polypeptide maybe purified from other cellular components using an affinity column,which binds to a tagged portion of the polypeptide. As is generallyknown in the art, it is believed that the order of conducting thevarious purification steps may be changed, or that certain steps may beomitted, and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptide within a fraction by SDS/PAGE analysis. Another method forassessing the purity of a fraction is to calculate the specific activityof the fraction, to compare it to the specific activity of the initialextract and to thus calculate the degree of purity. The actual unitsused to represent the amount of activity will, of course, be dependentupon the particular assay technique chosen to follow the purificationand whether or not the expressed protein or peptide exhibits adetectable activity. It is known that the migration of a polypeptide canvary, sometimes significantly, with different conditions of SDS/PAGE(Capaldi et al., 1977). It will therefore be appreciated that underdiffering electrophoresis conditions, the apparent molecular weights ofpurified or partially purified expression products may vary.

II. DISEASE STATES AND TREATMENT THEREOF

A. Cancer

While hyperproliferative diseases can be associated with any diseasewhich causes a cell to begin to reproduce uncontrollably, theprototypical example is cancer. One of the key elements of cancer isthat the cell's normal apoptotic cycle is interrupted and thus agentsthat interrupt the growth of the cells are important as therapeuticagents for treating these diseases. In this disclosure, the compounds ofthe present disclosure may be used to lead to decreased cell counts andas such can potentially be used to treat a variety of types of cancerlines. In some aspects, it is anticipated that the compounds of thepresent disclosure may be used to treat virtually any malignancy.

Cancer cells that may be treated with the compounds of the presentdisclosure include but are not limited to cells from the bladder, blood,bone, bone marrow, brain, breast, colon, esophagus, gastrointestine,gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate,skin, stomach, pancreas, testis, tongue, cervix, or uterus. In addition,the cancer may specifically be of the following histological type,though it is not limited to these: neoplasm, malignant; carcinoma;carcinoma, undifferentiated; giant and spindle cell carcinoma; smallcell carcinoma; papillary carcinoma; squamous cell carcinoma;lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;transitional cell carcinoma; papillary transitional cell carcinoma;adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; in situ pulmonaryadenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; sertoli cell carcinoma; Leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; malignantmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; Mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; paragranuloma; malignant lymphoma, smalllymphocytic; malignant lymphoma, large cell, diffuse; malignantlymphoma, follicular; mycosis fungoides; other specified non-Hodgkin'slymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma;immunoproliferative small intestinal disease; leukemia; lymphoidleukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cellleukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia;monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia;myeloid sarcoma; and hairy cell leukemia. In certain aspects, the tumormay comprise an osteosarcoma, angiosarcoma, rhabdosarcoma,leiomyosarcoma, Ewing sarcoma, glioblastoma, neuroblastoma, or leukemia.

B. Microbial Disease

In another aspect, the disclosure relates to certain infectiousdiseases. Infection is the invasion of an organism's body tissues bydisease-causing agents, their multiplication, and the reaction of hosttissues to the infectious agents and the toxins they produce. Infectiousdisease, also known as transmissible disease or communicable disease, isillness resulting from an infection. Microbial infections are thosecaused by microbes, i.e., viruses, bacteria, fungi and certainunicellular parasitic organisms. Ascomycota, including yeasts such asCandida, are of particular relevance. Cryptococcus neoformans, Candidaglabrata and Paracoccidioides lutzii also are known to use heme.

C. Combination Therapy

It is envisioned that the compounds of the present disclosure may beused in combination therapies with one or more other therapies or acompound which mitigates one or more of the side effects experienced bythe patient. It is common in the field of medicine to combinetherapeutic modalities. The following is a general discussion oftherapies that may be used in conjunction with the therapies of thepresent disclosure.

For example, to treat cancers or other disease using the methods andcompositions of the present disclosure, one would generally contact atumor cell or subject with a compound and at least one other therapy.These therapies would be provided in a combined amount effective toachieve a reduction in one or more disease parameter. This process mayinvolve contacting the cells/subjects with the both agents/therapies atthe same time, e.g., using a single composition or pharmacologicalformulation that includes both agents, or by contacting the cell/subjectwith two distinct compositions or formulations, at the same time,wherein one composition includes the compound and the other includes theother agent.

Alternatively, the compounds of the present disclosure may precede orfollow the other treatment by intervals ranging from minutes to weeks.One would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the therapies wouldstill be able to exert an advantageously combined effect on thecell/subject. In such instances, it is contemplated that one wouldcontact the cell with both modalities within about 12-24 hours of eachother, within about 6-12 hours of each other, or with a delay time ofonly about 1-2 hours. In some situations, it may be desirable to extendthe time period for treatment significantly; however, where several days(2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

It also is conceivable that more than one administration of either thecompound or the other therapy will be desired. Various combinations maybe employed, where a compound of the present disclosure is “A,” and theother therapy is “B,” as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B BBB/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/BBB B/A/B/B B/B/A/B

Other combinations are also contemplated. The following is a generaldiscussion of cancer therapies that may be used combination with thecompounds of the present disclosure.

1. Cancer

Chemotherapy.

The term “chemotherapy” refers to the use of drugs to treat cancer. A“chemotherapeutic agent” is used to connote a compound or compositionthat is administered in the treatment of cancer. These agents or drugsare categorized by their mode of activity within a cell, for example,whether and at what stage they affect the cell cycle. Alternatively, anagent may be characterized based on its ability to directly cross-linkDNA, to intercalate into DNA, or to induce chromosomal and mitoticaberrations by affecting nucleic acid synthesis. Most chemotherapeuticagents fall into the following categories: alkylating agents,antimetabolites, antitumor antibiotics, mitotic inhibitors, andnitrosoureas.

Examples of chemotherapeutic agents include alkylating agents such asthiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cyclophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin γ₁ and calicheamicin ω₁;dynemicin, including dynemicin A; uncialamycin and derivatives thereof;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores, aclacinomycins, actinomycin, authrarnycin,azaserine, bleomycins, cactinomycin, carubicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozotocin, tubercidin,ubenimex, zinostatin, or zorubicin; anti-metabolites such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogs such asdenopterin, methotrexate, pteropterin, trimetrexate; purine analogs suchas fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as folinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes(especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., paclitaxel and docetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumcoordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11);topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; capecitabine; cisplatin (CDDP),carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, busulfan,nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin,plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogenreceptor binding agents, taxol, paclitaxel, docetaxel, gemcitabine,navelbine, farnesyl-protein tansferase inhibitors, transplatinum,5-fluorouracil, vincristine, vinblastine and methotrexate andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Radiotherapy.

Radiotherapy, also called radiation therapy, is the treatment of cancerand other diseases with ionizing radiation. Ionizing radiation depositsenergy that injures or destroys cells in the area being treated bydamaging their genetic material, making it impossible for these cells tocontinue to grow. Although radiation damages both cancer cells andnormal cells, the latter are able to repair themselves and functionproperly.

Radiation therapy used according to the present disclosure may include,but is not limited to, the use of γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors induce a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 12.9 to 51.6 mC/kg for prolongedperiods of time (3 to 4 wk), to single doses of 0.516 to 1.55 C/kg.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

Radiotherapy may comprise the use of radiolabeled antibodies to deliverdoses of radiation directly to the cancer site (radioimmunotherapy).Antibodies are highly specific proteins that are made by the body inresponse to the presence of antigens (substances recognized as foreignby the immune system). Some tumor cells contain specific antigens thattrigger the production of tumor-specific antibodies. Large quantities ofthese antibodies can be made in the laboratory and attached toradioactive substances (a process known as radiolabeling). Once injectedinto the body, the antibodies actively seek out the cancer cells, whichare destroyed by the cell-killing (cytotoxic) action of the radiation.This approach can minimize the risk of radiation damage to healthycells.

Conformal radiotherapy uses the same radiotherapy machine, a linearaccelerator, as the normal radiotherapy treatment but metal blocks areplaced in the path of the x-ray beam to alter its shape to match that ofthe cancer. This ensures that a higher radiation dose is given to thetumor. Healthy surrounding cells and nearby structures receive a lowerdose of radiation, so the possibility of side effects is reduced. Adevice called a multi-leaf collimator has been developed and may be usedas an alternative to the metal blocks. The multi-leaf collimatorconsists of a number of metal sheets which are fixed to the linearaccelerator. Each layer can be adjusted so that the radiotherapy beamscan be shaped to the treatment area without the need for metal blocks.Precise positioning of the radiotherapy machine is very important forconformal radiotherapy treatment and a special scanning machine may beused to check the position of internal organs at the beginning of eachtreatment.

High-resolution intensity modulated radiotherapy also uses a multi-leafcollimator. During this treatment the layers of the multi-leafcollimator are moved while the treatment is being given. This method islikely to achieve even more precise shaping of the treatment beams andallows the dose of radiotherapy to be constant over the whole treatmentarea.

Although research studies have shown that conformal radiotherapy andintensity modulated radiotherapy may reduce the side effects ofradiotherapy treatment, it is possible that shaping the treatment areaso precisely could stop microscopic cancer cells just outside thetreatment area being destroyed. This means that the risk of the cancercoming back in the future may be higher with these specializedradiotherapy techniques.

Scientists also are looking for ways to increase the effectiveness ofradiation therapy. Two types of investigational drugs are being studiedfor their effect on cells undergoing radiation. Radiosensitizers makethe tumor cells more likely to be damaged, and radioprotectors protectnormal tissues from the effects of radiation. Hyperthermia, the use ofheat, is also being studied for its effectiveness in sensitizing tissueto radiation.

Immunotherapy.

In the context of cancer treatment, immunotherapeutics, generally, relyon the use of immune effector cells and molecules to target and destroycancer cells. Trastuzumab (Herceptin™) is such an example. The immuneeffector may be, for example, an antibody specific for some marker onthe surface of a tumor cell. The antibody alone may serve as an effectorof therapy or it may recruit other cells to actually affect cellkilling. The antibody also may be conjugated to a drug or toxin(chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussistoxin, etc.) and serve merely as a targeting agent. Alternatively, theeffector may be a lymphocyte carrying a surface molecule that interacts,either directly or indirectly, with a tumor cell target. Variouseffector cells include cytotoxic T cells and NK cells. The combinationof therapeutic modalities, i.e., direct cytotoxic activity andinhibition or reduction of ErbB2 would provide therapeutic benefit inthe treatment of ErbB2 overexpressing cancers.

In one aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist and any of these may be suitablefor targeting in the context of the present disclosure. Common tumormarkers include carcinoembryonic antigen, prostate specific antigen,urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68,TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor,laminin receptor, erb B and p155. An alternative aspect of immunotherapyis to combine anticancer effects with immune stimulatory effects. Immunestimulating molecules also exist including cytokines such as IL-2, IL-4,IL-12, GM-CSF, γ-IFN, chemokines such as MIP-1, MCP-1, IL-8 and growthfactors such as FLT3 ligand. Combining immune stimulating molecules,either as proteins or using gene delivery in combination with a tumorsuppressor has been shown to enhance anti-tumor effects (Ju et al.,2000). Moreover, antibodies against any of these compounds may be usedto target the anti-cancer agents discussed herein.

Examples of immunotherapies currently under investigation or in use areimmune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum,dinitrochlorobenzene and aromatic compounds (U.S. Pat. Nos. 5,801,005and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998),cytokine therapy, e.g., interferons α, β, and γ; IL-1, GM-CSF and TNF(Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998)gene therapy, e.g., TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Wardand Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945) andmonoclonal antibodies, e.g., anti-ganglioside GM2, anti-HER-2, anti-p185(Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311).It is contemplated that one or more anti-cancer therapies may beemployed with the gene silencing therapies described herein.

In active immunotherapy, an antigenic peptide, polypeptide or protein,or an autologous or allogenic tumor cell composition or “vaccine” isadministered, generally with a distinct bacterial adjuvant (Ravindranathand Morton, 1991; Morton et al., 1992; Mitchell et al., 1990; Mitchellet al., 1993). In adoptive immunotherapy, the patient's circulatinglymphocytes, or tumor infiltrated lymphocytes, are isolated in vitro,activated by lymphokines such as IL-2 or transduced with genes for tumornecrosis, and readministered (Rosenberg et al., 1988; 1989).

Checkpoint inhibitor therapy is another valuable therapy for combinationwith the compounds of the present disclosure. Checkpoint therapy targetsimmune checkpoints, key regulators of the immune system that whenstimulated can dampen the immune response to an immunologic stimulus.Some cancers can protect themselves from attack by stimulating immunecheckpoint targets. Checkpoint therapy can block inhibitory checkpoints,restoring immune system function. The first anti-cancer drug targetingan immune checkpoint was ipilimumab, a CTLA4 blocker approved in theUnited States in 2011. Currently approved checkpoint inhibitors targetthe molecules CTLA4, PD-1, and PD-L 1. PD-1 is the transmembraneprogrammed cell death 1 protein (also called PDCD1 and CD279), whichinteracts with PD-L1 (PD-1 ligand 1, or CD274). PD-L1 on the cellsurface binds to PD1 on an immune cell surface, which inhibits immunecell activity. Among PD-L1 functions is a key regulatory role on T cellactivities. It appears that (cancer-mediated) upregulation of PD-L1 onthe cell surface may inhibit T cells that might otherwise attack.Antibodies that bind to either PD-1 or PD-L1 and therefore block theinteraction may allow the T-cells to attack the tumor.

Surgery.

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative, andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent disclosure, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically controlled surgery(Mohs' surgery). It is further contemplated that the present disclosuremay be used in conjunction with removal of superficial cancers,precancers, or incidental amounts of normal tissue.

Upon excision of part or all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

In some particular embodiments, after removal of the tumor, an adjuvanttreatment with a compound of the present disclosure is believed to beparticularly efficacious in reducing the reoccurance of the tumor.Additionally, the compounds of the present disclosure can also be usedin a neoadjuvant setting.

Other Agents.

It is contemplated that other agents may be used with the presentdisclosure. These additional agents include immunomodulatory agents,agents that affect the upregulation of cell surface receptors and gapjunctions, cytostatic and differentiation agents, inhibitors of celladhesion, agents that increase the sensitivity of the hyperproliferativecells to apoptotic inducers, or other biological agents.Immunomodulatory agents include tumor necrosis factor; interferon α, β,and γ; IL-2 and other cytokines; F42K and other cytokine analogs; orMIP-1, MIP-1β, MCP-1, RANTES, and other chemokines. It is furthercontemplated that the upregulation of cell surface receptors or theirligands such as Fas/Fas ligand, DR4 or DR5/TRAIL (Apo-2 ligand) wouldpotentiate the apoptotic inducing abilities of the present disclosure byestablishment of an autocrine or paracrine effect on hyperproliferativecells. Increased intercellular signaling by elevating the number of gapjunctions would increase the anti-hyperproliferative effects on theneighboring hyperproliferative cell population. In other embodiments,cytostatic or differentiation agents may be used in combination with thepresent disclosure to improve the anti-hyperproliferative efficacy ofthe treatments. Inhibitors of cell adhesion are contemplated to improvethe efficacy of the present disclosure. Examples of cell adhesioninhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin.It is further contemplated that other agents that increase thesensitivity of a hyperproliferative cell to apoptosis, such as theantibody c225, could be used in combination with the present disclosureto improve the treatment efficacy.

There have been many advances in the therapy of cancer following theintroduction of cytotoxic chemotherapeutic drugs. However, one of theconsequences of chemotherapy is the development/acquisition ofdrug-resistant phenotypes and the development of multiple drugresistance. The development of drug resistance remains a major obstaclein the treatment of such tumors and therefore, there is an obvious needfor alternative approaches such as gene therapy.

Another form of therapy for use in conjunction with chemotherapy,radiation therapy or biological therapy includes hyperthermia, which isa procedure in which a patient's tissue is exposed to high temperatures(up to 41.1° C.). External or internal heating devices may be involvedin the application of local, regional, or whole-body hyperthermia. Localhyperthermia involves the application of heat to a small area, such as atumor. Heat may be generated externally with high-frequency wavestargeting a tumor from a device outside the body. Internal heat mayinvolve a sterile probe, including thin, heated wires or hollow tubesfilled with warm water, implanted microwave antennae, or radiofrequencyelectrodes.

A patient's organ or a limb is heated for regional therapy, which isaccomplished using devices that produce high energy, such as magnets.Alternatively, some of the patient's blood may be removed and heatedbefore being perfused into an area that will be internally heated.Whole-body heating may also be implemented in cases where cancer hasspread throughout the body. Warm-water blankets, hot wax, inductivecoils, and thermal chambers may be used for this purpose.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and general safety and purity standards as required byFDA's Division of Biological Standards and Quality Control of the Officeof Compliance and Biologics Quality.

2. Anti-Fungal Agents

Combination therapies with the HeSP's of the present disclosure andknown anti-fungal agents are also contemplated. Such agents includefluconazole (FLC), itraconazole (ITC), ketoconazole (KTC), posaconazole(POS), and voriconazole (VOR), ketoconazolev (KTC), undecylic acid(undecanoic acid), nystatin (NYS), naftifine (NAF), tolnaftate,amorolfine, butenafine (BTF), miconazole (MCZ), econazole, ciclopirox,oxiconazole, sertaconazole, efinaconazole, clotrimazole (CLO),sulconazole, tioconazole, tavaborole, terbinafine (TER), mancozeb,tricyclazole, carbendazim, hexaconazole, propineb, metalaxyl, benomyl(BEN) (Methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate),difenoconazole, propiconazole (PCZ), kitazin, tebuconazole (TER),tridemorph (TDM), and metconazole (MET).

III. FORMULATION AND ADMINISTRATION

In another aspect, for administration to a patient in need of suchtreatment, pharmaceutical formulations (also referred to as apharmaceutical preparations, pharmaceutical compositions, pharmaceuticalproducts, medicinal products, medicines, medications, or medicaments)comprise a therapeutically effective amount of a compound disclosedherein formulated with one or more excipients and/or drug carriersappropriate to the indicated route of administration. In someembodiments, the compounds disclosed herein are formulated in a manneramenable for the treatment of human and/or veterinary patients. In someembodiments, formulation comprises admixing or combining one or more ofthe compounds disclosed herein with one or more of the followingexcipients: lactose, sucrose, starch powder, cellulose esters ofalkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone,and/or polyvinyl alcohol. In some embodiments, e.g., for oraladministration, the pharmaceutical formulation may be tableted orencapsulated. In some embodiments, the compounds may be dissolved orslurried in water, polyethylene glycol, propylene glycol, ethanol, cornoil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodiumchloride, and/or various buffers. In some embodiments, thepharmaceutical formulations may be subjected to pharmaceuticaloperations, such as sterilization, and/or may contain drug carriersand/or excipients such as preservatives, stabilizers, wetting agents,emulsifiers, encapsulating agents such as lipids, dendrimers, polymers,proteins such as albumin, nucleic acids, and buffers.

Pharmaceutical formulations may be administered by a variety of methods,e.g., orally or by injection (e.g. subcutaneous, intravenous, andintraperitoneal). Depending on the route of administration, thecompounds disclosed herein may be coated in a material to protect thecompound from the action of acids and other natural conditions which mayinactivate the compound. To administer the active compound by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation. In some embodiments, the active compound may beadministered to a patient in an appropriate carrier, for example,liposomes, or a diluent. Pharmaceutically acceptable diluents includesaline and aqueous buffer solutions. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes.

The compounds disclosed herein may also be administered parenterally,intraperitoneally, intraspinally, or intracerebrally. Dispersions can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (such as,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

The compounds disclosed herein can be administered orally, for example,with an inert diluent or an assimilable edible carrier. The compoundsand other ingredients may also be enclosed in a hard or soft-shellgelatin capsule, compressed into tablets, or incorporated directly intothe patient's diet. For oral therapeutic administration, the compoundsdisclosed herein may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchpharmaceutical formulations is such that a suitable dosage will beobtained.

The therapeutic compound may also be administered topically to the skin,eye, ear, or mucosal membranes. Administration of the therapeuticcompound topically may include formulations of the compounds as atopical solution, lotion, cream, ointment, gel, foam, transdermal patch,or tincture. When the therapeutic compound is formulated for topicaladministration, the compound may be combined with one or more agentsthat increase the permeability of the compound through the tissue towhich it is administered. In other embodiments, it is contemplated thatthe topical administration is administered to the eye. Suchadministration may be applied to the surface of the cornea, conjunctiva,or sclera. Without wishing to be bound by any theory, it is believedthat administration to the surface of the eye allows the therapeuticcompound to reach the posterior portion of the eye. Ophthalmic topicaladministration can be formulated as a solution, suspension, ointment,gel, or emulsion. Finally, topical administration may also includeadministration to the mucosa membranes such as the inside of the mouth.Such administration can be directly to a particular location within themucosal membrane such as a tooth, a sore, or an ulcer. Alternatively, iflocal delivery to the lungs is desired the therapeutic compound may beadministered by inhalation in a dry-powder or aerosol formulation.

In some embodiments, it may be advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. In someembodiments, the specification for the dosage unit forms of theinvention are dictated by and directly dependent on (a) the uniquecharacteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofa selected condition in a patient. In some embodiments, active compoundsare administered at a therapeutically effective dosage sufficient totreat a condition associated with a condition in a patient. For example,the efficacy of a compound can be evaluated in an animal model systemthat may be predictive of efficacy in treating the disease in a human oranother animal.

In some embodiments, the effective dose range for the therapeuticcompound can be extrapolated from effective doses determined in animalstudies for a variety of different animals. In some embodiments, thehuman equivalent dose (HED) in mg/kg can be calculated in accordancewith the following formula (see, e.g., Reagan-Shaw et al., FASEB J.,22(3):659-661, 2008, which is incorporated herein by reference):HED(mg/kg)=Animal dose(mg/kg)×(Animal K _(m)/Human K _(m))

Use of the K_(m) factors in conversion results in HED values based onbody surface area (BSA) rather than only on body mass. K_(m) values forhumans and various animals are well known. For example, the K_(m) for anaverage 60 kg human (with a BSA of 1.6 m²) is 37, whereas a 20 kg child(BSA 0.8 m²) would have a K_(m) of 25. K_(m) for some relevant animalmodels are also well known, including: mice K_(m) of 3 (given a weightof 0.02 kg and BSA of 0.007); hamster K_(m) of 5 (given a weight of 0.08kg and BSA of 0.02); rat K_(m) of 6 (given a weight of 0.15 kg and BSAof 0.025) and monkey K_(m) of 12 (given a weight of 3 kg and BSA of0.24).

Precise amounts of the therapeutic composition depend on the judgment ofthe practitioner and are specific to each individual. Nonetheless, acalculated HED dose provides a general guide. Other factors affectingthe dose include the physical and clinical state of the patient, theroute of administration, the intended goal of treatment and the potency,stability and toxicity of the particular therapeutic formulation.

The actual dosage amount of a compound of the present disclosure orcomposition comprising a compound of the present disclosure administeredto a patient may be determined by physical and physiological factorssuch as type of animal treated, age, sex, body weight, severity ofcondition, the type of disease being treated, previous or concurrenttherapeutic interventions, idiopathy of the patient and on the route ofadministration. These factors may be determined by a skilled artisan.The practitioner responsible for administration will typically determinethe concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual patient. The dosage may beadjusted by the individual physician in the event of any complication.

In some embodiments, the therapeutically effective amount typically willvary from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kgto about 750 mg/kg, from about 100 mg/kg to about 500 mg/kg, from about1 mg/kg to about 250 mg/kg, from about 10 mg/kg to about 150 mg/kg inone or more dose administrations daily, for one or several days(depending of course of the mode of administration and the factorsdiscussed above). Other suitable dose ranges include 1 mg to 10,000 mgper day, 100 mg to 10,000 mg per day, 500 mg to 10,000 mg per day, and500 mg to 1,000 mg per day. In some embodiments, the amount is less than10,000 mg per day with a range of 750 mg to 9,000 mg per day.

In some embodiments, the amount of the active compound in thepharmaceutical formulation is from about 2 to about 75 weight percent.In some of these embodiments, the amount if from about 25 to about 60weight percent.

Single or multiple doses of the agents are contemplated. Desired timeintervals for delivery of multiple doses can be determined by one ofordinary skill in the art employing no more than routineexperimentation. As an example, patients may be administered two dosesdaily at approximately 12-hour intervals. In some embodiments, the agentis administered once a day.

The agent(s) may be administered on a routine schedule. As used herein aroutine schedule refers to a predetermined designated period of time.The routine schedule may encompass periods of time which are identical,or which differ in length, as long as the schedule is predetermined. Forinstance, the routine schedule may involve administration twice a day,every day, every two days, every three days, every four days, every fivedays, every six days, a weekly basis, a monthly basis or any set numberof days or weeks there-between. Alternatively, the predetermined routineschedule may involve administration on a twice daily basis for the firstweek, followed by a daily basis for several months, etc. In otherembodiments, the invention provides that the agent(s) may be takenorally and that the timing of which is or is not dependent upon foodintake. Thus, for example, the agent can be taken every morning and/orevery evening, regardless of when the patient has eaten or will eat.

IV. NON-THERAPEUTIC APPLICATIONS

A. Diagnostic

Zinc protoporphyrin (ZnPP) is a compound found in red blood cells whenheme production is inhibited by lead and/or by lack of iron. Instead ofincorporating a ferrous ion, to form heme, protoporphyrin IX, theimmediate precursor of heme, incorporates a zinc ion, forming ZPP. Thereaction to insert a ferrous ion into protoporphyrin IX is catalyzed bythe enzyme ferrochelatase. Measurement of zinc protoporphyrin in redcells has been used as a screening test for lead poisoning and for irondeficiency. There are a number of specific clinical situations in whichthis measurement has been found to be useful.

Zinc protoporphyrin levels can be elevated as the result of a number ofconditions, for instance lead poisoning, iron deficiency, sickle cellanemia, sideroblastic anemia, anemia of chronic disease, vanadiumexposure, erythropoietic protoporphyria, and varying types of cancer.Because zinc proptoporphyrin has fluorescence, the inventor tested itsbinding to HeSP's. When bound, the fluorescence intensity is increasedby about a hundred-fold. Thus, ZnPP bound with HeSP may be used todetect tumors in in vivo imaging or in biological samples for any of theother diseases described above.

B. Biofilm Treatment

Biofilms may form on a wide variety of surfaces, including livingtissues, indwelling medical devices, industrial or potable water systempiping, or natural aquatic systems. HeSP's can be used as cleaningagents, emulsifiers, dispersants, surfactants, or antifungal,antibiofilm, or antifouling agents to remove disease-causing organismsfrom external surfaces, including human and animal tissue such as skinand wounds. They can be used in different products such as soaps,detergents, deodorizers, stain removers, health and skincare products,cosmetics, antiseptics, and household, industrial, institutional, andclinical cleaners. They can also be used to remove algae, mold, orslime. HeSP's can be used alone, or in combination with otherantimicrobial or antifungal agents.

A spectrum of indwelling medical devices (e.g., ocular lenses, dentalimplants, central venous catheters and needleless connectors,endotracheal tubes, intrauterine devices, mechanical heart valves,coronary stents, vascular bypass grafts, pacemakers, peritoneal dialysiscatheters, prosthetic joints, central nervous system shunts,tympanostomy tubes, urinary catheters, and voice prostheses) or otherdevices used in the health-care environment have been shown to harborbiofilms, resulting in measurable rates of device-associated infections.

The HeSP's can be used on the surface of or within medical devices toprovide long term protection against microbial colonization and reducethe incidence of device-related infections. These substances can also beincorporated as an anti-biofilm forming agent, in combination with ananti-fungal, into coatings for indwelling medical devices, instruments,and other clinical surfaces. Coatings will sufficiently kill or inhibitthe initial colonizing organism and prevent device-related infection aslong as the substance is presented in an inhibitory concentration at thedevice-microbe interface.

The HeSP's, either administered alone or as part of a coating or medicaldevice, can reduce or prevent biofilms. In certain embodiments, biofilmsare reduced by about 1.0 log, about 1.5 logs, about 2.0 logs, about 2.5logs, about 3.0 logs, about 3.5 logs, about 4.0 logs, about 4.5 logs, orabout 5.0 logs, or by any number bound by the range of about 1.0 toabout 5.0 logs.

The medical devices which are amenable to coatings of the subjectanti-biofilm substances generally have surfaces composed ofthermoplastic or polymeric materials such as polyethylene, Dacron,nylon, polyesters, polytetrafluoroethylene, polyurethane, latex,silicone elastomers and the like. Devices with metallic surfaces arealso amenable to coatings with the anti-biofilm substances. Suchdevices, for example bone and joint prosthesis, can be coated by cementmixture containing the subject anti-biofilm substances. During implantuse, the anti-biofilm substances leach from the cement into thesurrounding prosthesis surface environment.

Various methods can be employed to coat the surfaces of medical deviceswith the anti-biofilm substances. For example, one of the simplestmethods would be to flush the surfaces of the device with a solution ofthe anti-biofilm substance. The flushing solution would normally becomposed of sterile water or sterile normal saline solutions. Anothermethod of coating the devices would be to first apply or adsorb to thesurface of the medical device a layer of tridodecylmethyl ammoniumchloride (TDMAC) surfactant followed by a coating layer of anti-biofilmsubstance. For example, a medical device having a polymeric surface,such as polyethylene, silastic elastomers, polytetrafluoroethylene orDarcon, can be soaked in a 5% by weight solution of TDMAC for 30 minutesat room temperature, air dried, and rinsed in water to remove excessTDMAC. Alternatively, TDMAC precoated catheters are commerciallyavailable; for example, arterial catheters coated with TDMAC areavailable from Cook Critical Care, Bloomington, Ind. The device carryingthe absorbed TDMAC surfactant coated can then be incubated in a solutionof the anti-biofilm substance for one hour or so, washed in sterilewater to remove unbound anti-biofilm substance and stored in a sterilepackage until ready for implantation. A further method useful to coatthe surface of medical devices with the subject antibiotic combinationsinvolves first coating the selected surfaces with benzalkonium chloridefollowed by ionic bonding of the anti-biofilm substance composition.Alternative methods and reagents provided in U.S. Pat. Nos. 4,107,121,4,442,133, 4,678,660 and 4,749,585, 4,895,566, 4,917,686, 4,952,419, and5,013,30, can be used to coat devices with the anti-biofilm substancesdisclosed herein.

The HeSP's can be directly incorporated into the polymeric matrix of themedical device at the polymer synthesis stage or at the devicemanufacture stage. An HeSP can also be covalently attached to themedical device polymer.

Biofilms in industrial systems cause severe clogging, contamination,corrosion, scale, slime, and biodeterioration. Microbial contaminationof the water distribution systems can occur if biofilms are sloughed offnaturally or removed by treatment. Biofilms in drinking water pipingsystems are also common. This results in decreased water quality andincreased treatment costs and health risks. Biofilms in pipes, fixturesand containers carrying water or other liquids cause reduced flow andincreased resistance to flow. Formation of biofilms on probes, sensors,screens and filters results in reduced efficiency. Microbial films thatgrow on the walls of heat exchanger tubes create additional heattransfer and fluid flow resistances. Formation of biofilms on ship hullsleads to biofouling resulting in increased fuel consumption and cleaningcosts. The food industry is also affected by the contamination caused bythese films which adhere easily to the walls of food processingequipment, and on the surface of food itself. Biofilms in cooling towersresults in reduced performance, degradation of material and alsoprovides a reservoir for pathogens. Building materials such as stone,bricks and concrete or clay based roof tiles, mortars and especially allnew materials for insulation and damming of humidity often containorganic compounds and are very susceptible to growth of sub-aerialbiofilms creating an anesthetic biopatina and reducing durability.Chemical and physical biodeteriorative forces, phenomena and processesfurther create damage on old and new buildings. Depending on theenvironmental conditions water retention and penetration the surfacebiofilms may transform into networks going deeper into the material.Biocide impregnation of new materials and biocide treatments ofmonuments create health and environmental hazards. Biofilm on surfacesalso captures pollutants, noxious particles, elements, spores, and othercontaminants.

Fouling is an undesirable growth of biological material on a surfaceimmersed in water. Fouling usually starts with adhering and spreading ofpopulations of microbes over surfaces that are in contact with water.Such structures may include: pilings, marine markers, underseaconveyances like cabling and pipes, fishing nets, bulkheads, coolingtowers, and any device or structure that operates submerged.

An HeSP can be incorporated into marine coatings to limit undesirablemarine fouling. The anti-fouling coatings of this disclosure offersignificant advantages over previous attempts to solve marine foulingproblems. The coatings disclosed herein can be formulated so as not tocontain toxic materials (such as heavy metals), and still retain theirefficacy. This avoids the environmental concerns associated with the useof heavy metal biocides.

In some embodiments, the methods of the present disclosure compriseapplying to a surface a composition comprising an HeSP effective toinhibit the growth of a biofilm on the surface. In some embodiments, thesurface is an indwelling medical device. In some embodiments, thesurface is a surface exposed to water. In some embodiments, the surfaceis a piece of industrial equipment.

V. EXAMPLES

The following Examples are intended to illustrate the above disclosureand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the disclosure could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the disclosure. The disclosure may be further understood bythe following non-limiting examples.

Example 1 Materials and Methods

Reagents.

Succinyl acetone was purchased from Sigma-Aldrich (Catalog #D1415-1G).Heme was purchased from Frontier Scientific Inc. (Catalog #H651-9). Zinc(II) protoporphyrin IX (Catalog #Zn 625-9) was purchased from FrontierScientific Inc. Tin (IV) protoporphyrin was purchased from PorphyrinProducts Inc (Catalog #Sn749-9). Deferoxamine mesylate was purchasedfrom Sigma-Aldrich (Cat #D9533-1G). Ferric chloride was purchased fromSigma-Aldrich (Catalog #157740-100G). D-Luciferin and the Opal 4 colorIHC kit were purchased from PerkinElmer (USA). [4-¹⁴C]-5-aminolevulinicacid was custom synthesized by PerkinElmer. Antibodies were purchasedfrom Santa Cruz Biotechnology, Cell Signaling Technology, NovusBiologicals, and abcam. HSP1 and HSP2 were purified with the pET11aexpression system. The pET11a expression vector for Y. pestis HasAresidues 1-193 was kindly provided by Dr. Mario Rivera (University ofKansas) (23). HSP1 contains the Q32H mutation, and HSP2 contains Q32HY75M double mutations. The mutations were generated with the QuikChangeII Site-Directed Mutagenesis Kit (Agilent Technologies). The doublemutations were generated with the expression vector for the Q32Hmutation. All DNA clones were confirmed by sequencing (Eurofins GenomicsLLC). HSP1 and HSP2 were purified with a Q-Sepharose Fast Flow column(GE Healthcare), followed by size exclusion chromatography, as described(23). The reporter plasmids for measuring subcellular heme levels werekindly provided by Dr. Iqbal Hamza (24).

Cell Culture and Analyses of Tumorigenic Functions.

HBEC30KT (RRID:CVCL_AS83) and HCC4017 (RRID:CVCL_V579) cell linesrepresenting normal, non-tumorigenic and NSCLC cells from the samepatient (25, 26), respectively, were provided by Dr. John Minna's lab(UT Southwestern Medical Center) as a gift. They were developed from thesame patient and were maintained in ACL4 medium supplemented with 2%heat-inactivated, fetal bovine serum (25). A pair of bronchialepithelial cell lines consisting of normal, non-tumorigenic cell lineNL20 (ATCC Cat #CRL-2503, RRID:CVCL_3756) and tumorigenic cell lineNL20-TA (ATCC Cat #CRL-2504, RRID:CVCL_3757), was purchased fromAmerican Type Culture Collection (ATCC). NL20 and NL20-TA cell lineswere maintained in Ham's F12 medium with 1.5 g/L sodium bicarbonate, 2.7g/L glucose, 2.0 mM L-glutamine, 0.1 mM nonessential amino acids, 0.005mg/ml insulin, 10 ng/ml epidermal growth factor, 0.001 mg/mltransferrin, 500 ng/ml hydrocortisone and 4% fetal bovine serum. Allother NSCLC cell lines, H1299 (ATCC Cat #CRL-5803, RRID:CVCL_0060), A549(ATCC Cat #CRM-CCL-185, RRID:CVCL_0023), H460 (ATCC Cat #HTB-177,RRID:CVCL_0459), Calu-3 (ATCC Cat #HTB-55, RRID:CVCL_0609), and H1395(ATCC Cat #CRL-5868, RRID:CVCL_1467) were purchased from ATCC,maintained in RPMI medium, and supplemented with 5% heat-inactivated,fetal bovine serum. All experiments using cells were conducted betweenpassages 3-5 from revival of the initial frozen stocks. experiments Celllines expressing luciferase were generated by infection with lentiviralparticles bearing the EF1α-Luciferase (firefly) gene (AMSBIO). Celllines were authenticated by Genetica and were found to be 96% identicalto the standard (authentication requires >80%). Cell lines were testedfor mycoplasma using a MycoFluor™ Mycoplasma Detection Kit (MolecularProbes), and the results were negative.

For generation of stable overexpression lines overexpressing ALAS1 orSLC48A1, lentiviral vectors expressing ALAS1, SLC48A1, and eGFP (controlvector) were purchased from Genecoepia. The expression vectors for ALAS1and SLC48A1 also express eGFP, making them comparable with the controland easy for verification of positive clones. All vectors carry theneomycin selectable marker. Lentivirus particles were generated byco-transfecting 293T cells with packaging plasmids pMD2.G (addgeneplasmid #12259, RRID:Addgene 12259) and psPAX2 (addgene plasmid #12260,RRID:Addgene_12260) and vector for ALAS1 or SLC48A1 using Lipofectamine3000. pMD2.G and psPAX2 were gifts from Didier Trono. For generatingstable overexpression cell lines, H1299 and A549 cells (70-80%confluent) were transduced with virus particles (2.8×10⁴ units/well) in48-well tissue culture plates. After series of passes and antibioticselection stable clones were selected and verified for overexpression byWestern blotting.

Cell proliferation was measured by detecting the luciferase activity inlive cells and by using a hemocytometer. Cell migration and invasionassays were carried out with BD Falcon cell culture inserts (CorningLife Sciences) and the manufacturer's cell migration, chemotaxis, andinvasion assay protocols. For the colony formation assay, 5000 NSCLCcells were seeded in every well in 6-well tissue culture plates intriplicates. Cells were treated with 0.5 mM succinyl acetone (SigmaAldrich), 10 μM HSPs, 50 μM deferoxamine mesylate (DFX), 10 μM zinc (II)protoporphyrin IX, 10 μM tin (IV) protoporphyrin IX, or 50 μM ferricchloride for 6 days. For heme add-back experiments, 10 μM heme wasincluded. Medium was changed every 3 days. After 6 days of treatment,cells were fixed with 70% ethanol and stained with 0.5% crystal violet.Images were acquired by using Carestream Gel Logic GL-112 imagingsystem.

Measurement of Heme Synthesis and Uptake.

Measurement of heme synthesis in cells was carried out exactly asdescribed (27, 28). Briefly, 0.3 μC [4-¹⁴C]-5-aminolevulinic acid (ALA)was added to each culture plate for 15 hours. Heme was subsequentlyextracted, and radiolabeled heme was quantified as described (29). Formeasuring heme uptake, a fluorescent analog of heme, zinc protoporphyrinIX (ZnPP), was used, as described previously (30, 31). Briefly, 10,000NSCLC cells were seeded in 96-well plates. Cells were incubated for 3hours with 60 μM ZnPP in the presence or the absence of 40 μM HSPs.Fluorescence intensity was measured with a Biotek Cytation 5 platereader. Experiments were conducted in triplicates, and ZnPP uptake wasnormalized with total cellular proteins. For measuring heme levels invarious organelles, the inventor used peroxidase-based reporters whichexpress peroxidase activity along with a fluorescent marker like mCherryor eGFP in each organelle (24). Heme levels were measured exactly asdescribed and normalized with the fluorescent signals to correct forvariations, such as that in transfection efficiency. Only Calu-3 did notshow sufficient fluorescent signals to allow proper measurements (24).

Measurement of Oxygen Consumption and ATP Levels.

Oxygen consumption was measured, as described previously (16). Briefly,10⁶ cells (in 350 μl) were introduced into the chamber of an Oxygraphsystem (Hansatech Instruments), with a Clark-type electrode placed atthe bottom of the respiratory chamber. During measurements, the chamberwas thermostated at 37° C. by a circulating water bath. Anelectromagnetic stir bar was used to mix the contents of the chamber.

Total ATP was measured with the ATP-determination kit (Molecular Probes)following the manufacturer's protocol. Briefly, cultured cells werecollected and immediately placed in ice-cold lysis buffer (CellSignaling) with protease and phosphatase inhibitors. Cell lysates werethen centrifuged at 10,000 g for 10 min. 10 μl of lysates or 10 μl ofATP standard solution was added to 90 μl of reaction buffer in each wellof a 96-well plate. Luminescence was measured using a Biotek Cytation 5plate reader. All experiments were carried out in triplicate, and thebackground luminescence was subtracted from the measurement. ATPconcentrations were calculated from the ATP standard curve andnormalized with the numbers of cells used. To measure oxygen consumptionrates and ATP levels from freshly isolated tumors, subcutaneous tumorswere surgically resected from mice and cut into small pieces. Tumorswere weighed and homogenized immediately using mechanical homogenizer togain a homogenous cell suspension. Tissue debris was removed by gentlecentrifugation. Cells were suspended in 400 ul of complete medium, andOCR was measured using a Clark-type electrode. ATP levels were measuredwith an ATP determination kit (Molecular probes). Both Oxygenconsumption rates and ATP levels were normalized with protein amounts.

Preparation of Protein Extracts and Western Blotting.

Lung non-tumorigenic and tumorigenic cells were maintained (passage3-5), collected, and lysed by using the RIPA buffer (Cell SignalingTechnology) containing the protease inhibitor cocktail. Proteinconcentrations were determined by using the BCA assay kit (ThermoScientific). 50 μg of proteins from each treatment condition wereelectrophoresed on 10% SDS-polyacrylamide gels, and then transferredonto the Immuno-Blot PVDF Membrane (Bio-Rad). The membranes were probedwith antibodies, followed by detection with a chemiluminescence Westernblotting kit (Roche Diagnostics). The signals were detected by using aCarestream image station 4000MM Pro, and quantitation was performed byusing the Carestream molecular imaging software version 5.0.5.30(Carestream Health, Inc.). Antibodies used include those to thefollowing proteins: ALAS1 (1:1000, Novus Cat #NBP1-91656,RRID:AB_11048622), SLC46A1 (1:1000, Santa Cruz Biotechnology Cat#sc-134997, RRID:AB_11149379), SLC48A1 (1:1000, Santa Cruz BiotechnologyCat #sc-101957, RRID:AB_2191218), CYCS (1:1000, Santa Cruz BiotechnologyCat #sc-7159, RRID:AB_2090474), COX4 (1:1000, Santa Cruz BiotechnologyCat #sc-292052, RRID:AB_10843648), NRF1 (1:1000 Cell SignalingTechnology Cat #46743, RRID: AB_2732888), TFAM (1:1000, Cell SignalingTechnology Cat #8076S, RRID:AB_10949110), TFRC (1:1000, Novus Cat#NB100-92243, RRID; AB_1216384), SLC40A1 (1:1000, Novus Cat #NBP1-21502,RRID:AB_2302075), and β-actin (1:1000, Cell Signaling Technology Cat#4967, RRID:AB_330288).

Animals.

NOD/SCID mice (IMSR Cat #CRL:394, RRID:IMSR_CRL:394) were purchased fromCharles River Laboratories. Mice were bred and cared for in a Universityof Texas at Dallas specific pathogen-free animal facility in accordancewith NIH guidelines. All animal procedures were conducted underprotocols approved by Institutional Animal Care and Use Committee(IACUC) at the University of Texas at Dallas. Animals were regularlyexamined for any signs of stress and euthanized according to presetcriteria.

Treatment of Human Xenograft Lung Tumors in NOD/SCID Mice.

To generate mice with NSCLC tumors in the lungs, 0.75×10⁶ H1299-luccells in serum-free medium were injected via tail vein in 6-8-week-oldfemale NOD/SCID mice. Alternatively, 0.75×10⁶ H1299-luc cells (passages3-5) in serum-free medium containing 50% Matrigel were implanteddirectly on the lung. Mice were anesthetized with 2.5% isoflurane andoxygen mixture. H1299-luc cells were injected via tail vein or wereinjected about 1.5 cm above the lower left rib line through theintercostal region. Mice were then placed on a heating pad and observeduntil they revived from anesthesia. Mice were randomized into threegroups (n=6 per group) that received vehicle (for control) or HSP2 (10mg/kg, I.V., every 3 days) or HSP2 (25 mg/kg, I.V., every 3 days).Treatments started post cell implantation when lung tumors weredetectable using bioluminescence imaging. Body weights were recordedonce every week. Treatments were started only after BLI detectedauthentic signals (>5×10⁶ photons/second) to ensure the properimplantation of tumors. Treatments were stopped, and mice weresacrificed after the untreated mice with tumors appeared moribund. Fordetecting the toxicity of HSP2 treatment on blood and liver functions,blood was obtained from these mice via the sub-mandibular vein beforesacrifice and was collected in blood collection tubes (BD microtainertubes Cat #365963 and Cat #365974 from Fischer scientific). Serumsamples were prepared and then used for determining hemoglobin levelswith a hemoglobin assay kit from Sigma-Aldrich (Cat #MAK115-1KT) and ALTlevels (alanine transaminase levels) with an ALT activity assay kit fromSigma-Aldrich (MAK052-1KT), respectively. Whole blood samples were usedfor counting red blood cells (RBCs) using a hemocytometer. Nomorphological differences were observed in red cells from treated anduntreated mice.

For subcutaneous tumor models, 2×10⁶ H1299-luc cells or H1299 cellsoverexpressing eGFP (Control) or ALAS1 or SLC48A1 in serum-free mediumcontaining 50% Matrigel were injected subcutaneously into the left flankregion of 4-6 weeks old female NOD/SCID mice. Mice were randomized intotreatment groups that received saline (for control) and HSP2 (I.V. 25mg/kg every 3 days), respectively. Body masses were recorded once everyweek. Treatments were started only after BLI detected authentic tumorsignals and tumors were visible to ensure the proper implantation oftumors. When the tumors in the control group reached 1 cm³, mice wereeuthanized by cervical dislocation. Tumors were resected and weighed.

In Vivo Bioluminescence Imaging (BLI).

Mice bearing lung tumor xenografts were imaged with an IVIS Lumina IIIIn Vivo Imaging system (Perkin Elmer). Briefly, mice were anesthetizedin the isoflurane chamber (2% isoflurane and oxygen), and luciferin(potassium salt; Perkin Elmer; 80 μl of 40 mg/ml) was administeredsubcutaneously between the scapulae. A BLI time course was acquired over30 mins (Exposure time: auto, F Stop: 1.2, Binning: medium). The imageswere quantified using Living Image software version 4.5.2 (PerkinElmer). Regions of interest (ROIs) were selected, and bioluminescencesignals between 600 to 60000 counts were accepted as authentic signals.The total bioluminescent signals (photon/sec) from ROIs of mice werecalculated as specified by the manufacturer's instructions.

Hematoxylin and Eosin (H&E) Staining.

Following the final imaging, mice were sacrificed. Lung tumors wereexcised and tumor tissues were prepared for histology. Paraffinembedding was performed at Histology core at University of TexasSouthwestern Medical Center. The paraffin blocks were sectioned toobtain 5 μm sections which were utilized for Hematoxylin and Eosinstaining. For H&E staining, tumor tissues were fixed in 4% formalin,embedded in paraffin and sectioned (5 μm). Then, sections were stainedwith H&E. Slides were scanned at a 40× resolution with an Olympus VS120slide scanner and quantified using Cell Sens software from Olympus.

Immunohistochemistry (IHC).

IHC was carried out exactly as described (32). Paraffin-embedded tumortissues from mice described above were used. Six independent sets ofhuman NSCLC grade 2 & 3 tissues and six independent sets of normal humanlung tissues in paraffin slides were purchased from US Biomax, Inc.(Rockville, Md.). Slides were deparaffinized, hydrated, and washed.After antigen retrieval, slides were blocked with 1×TBS/10% goat serum(16210-072, Gibco). Primary antibodies were diluted in 1×TBS/1% BSA/10%goat serum. The dilutions were 1:200 for ALAS1 (Santa Cruz BiotechnologyCat #sc-50531, RRID:AB_2225629), 1:200 for SLC48A1 (Santa CruzBiotechnology Cat #sc-101957, RRID:AB_2191218); 1:200 for CYCS (SantaCruz Biotechnology Cat #sc-7159, RRID:AB_2090474), 1:200 for PTGS2(Santa Cruz Biotechnology Cat #sc-7951, RRID:AB_2084972); 1:200 for TFAM(Cell Signaling Technology Cat #8076S, RRID:AB_10949110); COX4I1 1:200,(Santa Cruz Biotechnology Cat #sc-292052, RRID:AB_10843648), NRF1(1:1000 Cell Signaling Technology Cat #46743, RRID: AB_2732888); UQCRC21:100 (Santa Cruz Biotechnology Cat #sc-390378 RRID:AB_2754980) andCOX5A 1:100 (Abcam Cat #ab110262 RRID:AB_10861723). Sections wereincubated with primary antibodies overnight at 4° C. and then incubatedwith horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG(Thermo Fisher Scientific Cat #31460, RRID: AB_228341) at a dilution of1:200 in 1×TBS/1% BSA for 45 mins at room temperature (RT). Slides werestained with tyramide signal amplification (TSA)-conjugatedfluorophores, which were diluted 1:100 in 1× Plus Amplification Diluent(NEL810001KT, PerkinElmer). TSA-conjugated fluorophores were aspiratedand slides were then washed. DAPI, diluted in TBST, was added to slidesand incubated for 5 min at RT. Coverslips were mounted over the slidesusing VECTASHIELD mounting medium (Vector Laboratories), sealed, andstored in darkness at −20° C.

Slides were scanned at a 40× resolution with an Olympus VS120 slidescanner and quantified using cell Sens software from Olympus. DAPI wasused to visualize nuclei. Multiple regions of interest (ROIs) of equalarea were drawn over tumor regions. ROIs were selected, so that equalnumbers of cells (identified via nuclei) were included in each ROI. TheROIs were positioned evenly throughout tumor regions. ROIs were retestedunder three different filters—FITC, Cy3, and Cy5—to ensure that noartifacts were present. ROIs were re-positioned if artifacts werepresent under one or more filters. Minimum and maximum thresholds wereset to avoid any background signal. Mean signal intensity from all ROIswere averaged, and the corresponding negative control average wassubtracted to yield the signal intensity for each antigen.

Statistical Analyses of Data.

Data from different treatment groups of cells, mice, and tissues werecompared, and statistical analysis was performed with a Welch 2-samplet-test. For calculating correlation coefficients, the inventor used thePearson formula for calculating correlation coefficient r and p-value.

${r\left( {X,Y} \right)} = \frac{{\Sigma\left( {x - \overset{\_}{x}} \right)}\left( {y - \overset{\_}{y}} \right)}{\sqrt{{\Sigma\left( {x - \overset{\_}{x}} \right)}^{2}{\Sigma\left( {y - \overset{\_}{y}} \right)}^{2}}}$$p = \frac{r\sqrt{n - 2}}{\sqrt{1 - r^{2}}}$

Results

Heme Synthesis and Uptake are Elevated to Heterogeneous Degrees inSeveral Types of NSCLC Cell Lines, Leading to Elevated MitochondrialHeme Levels.

To gain insights into the degree of elevation and heterogeneity of hememetabolism and flux in lung tumors, the inventor measured hemebiosynthesis, uptake, and degradation in several representative types ofNSCLC cell lines. These include H1299 (with Nras Q61K p53 null), A549(with Kras G12S, LKB1 Q37*), H460 (with Kras K61H LKB1 Q37*), Calu-3(with Kras G13D p53 M237I mutations), and H1395 (with LKB1 deletion).She also used two pairs of cell lines representing normal lungepithelial cells (HBEC30KT and NL20 in FIGS. 1A-F, 2A-D, S1A-C & S2A-H)and tumorigenic cell lines (NSCLC line HCC4017 and NL20-TA in FIGS.1A-F, 2A-D, S1A-C & S2A-H) (25). Clearly, heme biosynthesis (FIG. 1A)and uptake (FIG. 1B) were both increased in NSCLC cell lines andNL20-TA, although the increases varied considerably among different celllines. When the folds of increase in heme biosynthesis and uptake wereadded for every cell line (FIG. 1C), they varied from 2- to 8-fold amongdifferent lung tumor cell lines. Increases in heme biosynthesis anduptake correlated with increases in the rate-limiting heme biosynthesisenzyme ALAS1 (FIG. S1A; r=0.90, p-value=0.0003) and the cell membraneheme uptake protein SLC46A1 (SLC46A1) (FIG. S1B; r=0.70, p-value=0.02),respectively.

Heme degradation was also elevated in NSCLC cell lines relative tonon-tumorigenic cell lines, albeit to a varying degree (FIG. 1D). Thisincrease correlated with the increase in HMOX1 enzyme (FIG. S1C; r=0.70;p-value=0.02). Iron is an essential nutrient and is closely linked toheme (33). Heme synthesis in non-erythroid cells is generally notaffected by iron (34). Nonetheless, the inventor detected the levels oftransferrin receptor (TFRC), which is responsible for cellular ironuptake from the circulation (35). She found that TFRC levels wereincreased in some, while unaffected or decreased in other NSCLC celllines (FIG. 1E). This is consistent with the idea that iron availabilityis not a limiting factor in NSCLC cells.

To determine how elevated heme metabolism in NSCLC cells affectssubcellular heme levels, the inventor used a series of previouslydeveloped subcellular peroxidase reporters designed to detectsubcellular heme levels in mitochondria, cytosol, nuclei, and otherorganelles (24). All lung cell lines were efficiently transfected withthe reporter plasmids, except for Calu-3, which did not allow efficienttransfection of reporter plasmids. Clearly, the mitochondrial hemelevels in NSCLC cell lines and the tumorigenic NL20-TA cell line wereelevated relative to non-tumorigenic cell lines (FIG. 1F). The increasein heme synthesis and uptake was correlated with the increase inintracellular mitochondrial heme levels: r=0.68, p-value=0.03. Hemelevels in other organelles were also increased in some tumor cell lines,but increases were not uniform (FIGS. S2A-E). The increase in hemesynthesis and uptake was not correlated significantly with heme levelsin other organelles. Mitochondrial heme is crucial for OXPHOS formationand function. These data suggest that increased heme synthesis anduptake in NSCLC cells leads to elevated mitochondrial heme levels.

Elevated Mitochondrial Heme Levels Lead to Intensified OxygenConsumption and ATP Generation in NSCLC Cell Lines.

Next, the inventor measured a series of bioenergetic and tumorigenicparameters. The rates of oxygen consumption (FIG. 2A) and levels ofintracellular ATP (FIG. 2B) were elevated in tumorigenic cell lines,except for Calu-3, relative to non-tumorigenic cell lines. Elevatedoxygen consumption should be accompanied by increased levels ofmitochondrial OXPHOS enzymes. Increased expression of mitochondrialproteins should be facilitated by regulators promoting mitochondrialbiogenesis, such as NRF1 and TFAM (36). Indeed, the levels of cytochromec (CYCS) and COX4I1 (subunits of OXPHOS complexes), as well as thehemoprotein PTGS2, were elevated in NSCLC cell lines relative tonon-tumorigenic cell lines (FIGS. S2F-H). Notably, two importantregulators promoting mitochondrial biogenesis, NRF1 and TFAM (FIGS. 2C &2D), were also upregulated in tumorigenic cell lines relative tonon-tumorigenic cells lines. Thus, these observations are consistentwith increased oxygen consumption rates and ATP levels in tumorigeniccell lines.

Measurements of migration (FIG. S3A) and invasion (FIG. S3B) in NSCLCcell lines showed that they exhibit varying degrees of tumorigenicity.Interestingly, the inventor found that the invasive capabilities ofNSCLC cell lines, oxygen consumption rates, and intracellular ATP levelswere well correlated with mitochondrial heme levels. The correlationcoefficients are as follows: mitochondrial heme and oxygen consumptionrates: Pearson r=0.72, p-value=0.02; mitochondrial heme and ATP levels:r=0.78, p-value=0.01; and mitochondrial heme and invasion: r=0.71,p-value=0.05. Together, these results strongly suggest that elevatedheme biosynthesis and uptake in NSCLC cell lines lead to elevated levelsof mitochondrial heme and OXPHOS subunits, which cause intensifiedoxygen consumption, ATP generation, and tumorigenic capabilities inNSCLC cells.

The inventor further confirmed the importance of enhanced levels ofproteins/enzymes relating to heme function and mitochondrial respirationin lung cancer. FIGS. S4A-B show that the levels of the rate-limitingheme synthetic enzyme ALAS1 and the heme transporter SLC48A1 (SLC48A1)were both significantly enhanced in human NSCLC tissues relative tonormal tissues. In the same vein, the heme-containing cytochrome c (FIG.S4C) and cyclooxygenase-2 (PTGS2) (FIG. S4D) were enhanced in humanNSCLC tissues relative to normal tissues. Both cytochrome c (CYCS) andPTGS2 levels have previously been shown to be elevated in NSCLC celllines and xenograft tumors (16). Notably, the levels of themitochondrial biogenesis regulator TFAM were also enhanced in humanNSCLC tissues (FIG. S4E), as is the case in NSCLC cell lines (FIG. 2D).Together, data from human NSCLC tissues, NSCLC cell lines, and xenografttumors show that proteins/enzymes relating to heme function andmitochondrial respiration are upregulated in NSCLC cells and tumors.

Engineered Heme-Sequestering Peptides (HSPs) can Inhibit Heme Uptake inNSCLC Cell Lines.

If elevated heme metabolism is crucial for the tumorigenic functions ofNSCLC cells, limiting heme availability may be effective for suppressinglung tumor growth and progression. Previous studies have identifiedsuccinyl acetone as an effective inhibitor of heme biosynthesis, as itinhibits the rate-limiting heme synthesis enzyme 5-aminolevulic synthase(ALAS1) in non-erythroid cells (37). However, succinyl acetone is notvery effective in suppressing lung tumors in mice (FIG. S5A). Therefore,the inventor tried to lower heme availability by taking advantage ofbacterial hemophores. The inventor took advantage of thewell-characterized Yersinia pestis hemophore HasA (23). She usedstructural comparisons of known HasAs and a computational algorithmbased on coevolution (38) to identify residues whose mutations may alterbut not disrupt heme-binding properties. She designed severalheme-sequestering peptides (HSPs), including HasA Q32H (HSP1) and HasAQ32H Y75M (HSP2) (FIG. S5B). These two peptides bind to heme strongly(FIG. S5C), like the wild-type HasA (23). The changed amino acids inHSP1 and HSP2 are known to coordinate heme well. Thus, the changes arenot expected to reduce heme binding. Interestingly, HSP1 and HSP2 haveenhanced capabilities to inhibit heme uptake in NSCLC cells (FIGS.3A-C). HSP2 is the most potent in inhibiting heme uptake by NSCLC celllines, reducing heme uptake by 5-fold in some cell lines (FIG. 3A).Furthermore, the effect of HSPs on heme uptake was reversed if more ZnPP(a heme analogue used for measuring heme uptake) was included (compare1×ZnPP, 2×ZnPP, and 2×ZnPP+HSP2 in FIG. 3A), indicating that HSP2 doesnot reduce heme uptake by causing other toxicities. Note that 1×ZnPPlikely saturated the capabilities of cells to uptake heme/ZnPP so that2×ZnPP did not cause more uptake.

As a bacterial hemophore, HasA is not internalized by human host cells.Thus, HSPs are not expected to be internalized by NSCLC cells. Indeed,HSP2 remained in the medium even after prolonged incubation with NSCLCcells (FIGS. S5D-E). Fluorescent images of NSCLC cells also showed thatZnPP-HSP2 did not enter cells (FIG. S5F), while ZnPP in the mediumwithout HSP2 entered cells and co-localized with mitotracker (FIG. S5G).Furthermore, the inventor detected the effect of HSP2 treatment onmitochondrial heme levels in NSCLC cells, because mitochondrial hemelevels are correlated with heme synthesis and uptake, as well asinvasion (see above results). FIG. 3B shows that mitochondrial hemelevels gradually decreased as the treatment time with HSP2 increased.Together, these results strongly suggest that HSP2 acts on NSCLC cellsby lowering heme uptake and mitochondrial heme levels.

HSPs effectively suppress NSCLC cell proliferation and tumorigenicfunctions. As expected, both HSP1 and HSP2 inhibited NSCLC cellproliferation in various NSCLC cell lines (FIG. 3C) and in adose-dependent manner (FIGS. S6A-D). The effects of HSPs on theproliferation of the HBEC30KT cell line representing normal lungepithelial cells were much less severe relative to NSCLC cell lines(FIG. 3C and FIG. S6A). This is consistent with the idea that normalcells do not need as much heme as NSCLC cells need. The inventor alsotested and compared the efficacies of HSP1, HSP2, and succinyl acetoneat inhibition of tumorigenic functions in NSCLC cells. Evidently, HSP2was more effective than succinyl acetone and HSP1 at inhibitingmigration of H1299 (FIG. 4A) and A549 cells (FIG. S7A). Likewise, HSP2was more effective than succinyl acetone and HSP1 at inhibiting invasionby H1299 (FIG. 4B) and A549 cells (FIG. S7B). Notably, addition of hemelargely reversed the effects of HSP1 and HSP2, like succinyl acetone(SA), on reducing proliferation, migration and invasion of NSCLC cells(FIG. 3C, FIGS. 4A-B, FIGS. S7A-B). The reversal of HSP1 and HSP2effects by heme addition supports the idea that the effects of HSP1 andHSP2, like SA, on migration and invasion are attributable to theireffect on heme uptake. The inventor also found that SA, HSP1, and HSP2strongly suppressed colony formation in H1299 (see FIGS. 4C-E) and A549(FIGS. S7C-E) cells. Inhibition of heme degradation by SnPP appeared toreduce colony formation in NSCLC H1299 (FIG. 4F) and A549 (FIG. S7F)cells.

Addition of heme to cells treated with SA, HSP1, or HSP2 largelyreversed the effects of these agents on colony formation (FIGS. 4C-E &FIGS. S7C-E), indicating that their effects are attributable to lack ofheme. As expected, iron chelator deferoxamine (DFX) also reduced colonyformation in NSCLC cells, and addition of iron largely reversed theeffect of DFX (FIG. 4G & FIG. S7G). Addition of heme to DFX-treatedcells partially reversed the effect on colony formation (FIG. 4G & FIG.S7G), but addition of iron to SA-, HSP1-, or HSP2-treated cells did notreverse the effects on colony formation (FIGS. 4C-E & FIGS. S7C-E). Thisis consistent with the fact that iron can be obtained via heme. However,extra iron cannot overcome the effect on heme synthesis or uptake,likely because iron is not a limiting factor in NSCLC cells, as in mostnon-erythroid cells (34). Consistent with this observation ofdifferential effects of iron and heme on colony formation, data in FIGS.S7H-I show that HSP2, unlike DFX, had no significant effects on thelevels of transferrin receptor TFRC and ferroportin SLC40A1.

HSP2 Effectively Suppresses the Growth of Human Tumor Xenografts inMice.

To further assess the anti-tumor activity of HSP2 in vivo, the inventorexamined the effects of administering HSP2 on the growth of humanxenograft tumors in the lungs of NOD/SCID mice (FIGS. 5A-H). Detectionof tumor growth and progression with BLI showed that HSP2 significantlysuppressed lung tumor growth and progression (FIGS. 5A & 5B). HSP2 didnot significantly change the body masses (FIG. 5C). Histologicalanalysis with H&E staining confirmed that 25 mg/kg of HSP2 nearlyeradicated the lung tumors (FIG. 5D). The inventor found that HSP2 waseffective at suppressing tumor growth when it was administered toNOD/SCID mice with larger tumor xenografts in the lung (FIGS. S8A-B;when treatments started the tumors used in FIG. S8 showed 10× higher BLIsignals than those in FIGS. 5A-H). HSP2 did not significantly affect redcell counts (FIG. S8C) and hemoglobin levels (FIG. S8D) in the blood, aswell as liver function shown by Alanine transaminase (ALT) activity(FIG. S8E). These results show that inhibition of heme uptake by HSP2can effectively suppress lung tumor growth and progression.

The inventor found that the levels of subunits of OXPHOS complexes,including COX5A (FIG. 5E), COX4I1 (FIG. 5F), UQCRC2 (FIG. 5G), and CYCS(FIG. 5H), were significantly reduced in HSP2-treated tumors, indicatingreduced oxygen consumption. To further ascertain the effect of HSP2 onoxygen consumption, the inventor decided to directly detect oxygenconsumption and ATP generation in lung tumors in mice. However, shefound that it is difficult to isolate sufficient populations of tumorcells from orthotopic lung tumors or do measurements quick enough tocollect valid data. To overcome these difficulties, she usedsubcutaneously implanted NSCLC tumors. HSP2 was very effective insuppressing subcutaneously implanted NSCLC tumors (FIGS. 6A-C). Notably,the oxygen consumption rates and ATP levels in HSP2-treated tumors wereboth significantly reduced relative to untreated tumors (populations ofcells isolated quickly from tumors) (FIGS. 6E-F).

Overexpression of ALAS1 or SLC48A1 Promotes Oxygen Consumption, ATPGeneration, Tumorigenic Functions of NSCLC Cells and Tumor Growth.

To further ascertain the importance of heme in promoting NSCLC tumors,the inventor generated NSCLC cell lines that overexpress therate-limiting heme synthesis enzyme ALAS1 or the heme uptakeprotein/transporter SLC48A1 (FIGS. 7A-H). She confirmed that relative tocontrol cells, cells overexpressing ALAS1 exhibited elevated hemesynthesis (FIG. 7A & FIG. S8F) while cells overexpressing SLC48A1exhibited elevated heme uptake (FIG. 7B & FIG. S8G). These cells alsoshowed elevated oxygen consumption (FIG. S8H). Importantly, these cellsoverexpressing ALAS1 or SLC48A1 exhibited enhanced migration (FIG. 7C),invasion (FIG. 7D), and colony formation (FIG. S8I). When these cellswere implanted subcutaneously in NOD/SCID mice, they form bigger tumorsthan control cells (FIGS. 7E-F). Furthermore, tumors overexpressingALAS1 or SLC48A1 exhibited elevated levels of oxygen consumption (FIG.7G) and ATP generation (FIG. 7H). Taken together, these results stronglysupport the idea that increased heme availability resulting fromelevated heme synthesis or uptake leads to higher oxygen consumption andATP generation, which in turn fuels NSCLC cell tumorigenic functions andtumor growth.

Discussion

In the 1920s, Otto Warburg demonstrated that tumor cells metabolizeglucose and generate lactate at higher levels than normal cells despitethe presence of ample oxygen, a phenomenon called the Warburg effect(39). However, elevated glucose consumption and glycolysis in tumorcells do not necessarily lead to diminished oxidative metabolism andOXPHOS (8, 9). Numerous previous studies have shown that high glycolyticrates occur concomitantly with oxidative phosphorylation (OXPHOS) incells of most tumors, and that function of mitochondrial OXPHOS isintact in most tumors (for a review, see (40)). More recent studies havedemonstrated the importance of mitochondrial OXPHOS in the growth andprogression of several types of tumors (41-43). Further, several studiesdemonstrated that oxidative metabolism and OXPHOS are crucial forconferring drug resistance of cancer cells and cancer stem cells. Fargeet al. showed that OXPHOS contributes to acute myeloid leukemiaresistance to cytarabine (13). Kuntz et al. showed that targetingmitochondrial OXPHOS eradicates drug-resistant CML stem cells (14). Leeet al. showed that MYC and MCL1 confer chemotherapy resistance byincreasing mitochondrial OXPHOS in cancer stem cells in triple negativebreast cancer (15).

Heme is a central molecule in mitochondrial OXPHOS and in virtually allprocesses relating to oxygen transport, storage, detoxification, andutilization (17, 18). Heme serves as an essential prosthetic group orcofactor for many proteins and enzymes that bind and use oxygen, such ascytochrome P450 and nitric oxide synthases (NOSs), and that detoxifyROSs, such as catalase and peroxidases. Three OXPHOS complexes, II, III,and IV, require heme for proper functioning. Multiple subunits incomplexes III and IV require heme as a prosthetic group, and differentforms of heme are present (40). Furthermore, heme serves as a signalingmolecule that directly regulates diverse processes, including theexpression and assembly of OXPHOS complexes (18, 19). Conversely, hemesynthesis occurs in mitochondria and requires oxygen (34). Thus, hemeand mitochondrial biogenesis are linked and are inter-dependent.Previously, the inventor showed that the levels of the rate-limitingheme biosynthetic enzyme ALAS1, heme uptake and transport proteinsSLC48A1 and SLC46A1, and oxygen-utilizing hemoproteins, including CYP1B1and PTGS2, are highly elevated in NSCLC tumors (16, 44). Other studiesalso showed that the expression of proteins involved in mitochondrialrespiration and heme function are elevated in the tumor tissues of NSCLCpatients (45, 46). Additionally, epidemiological studies indicated apositive association between intake of heme from meat and lung cancer(47).

Here, the inventor showed that the levels of heme biosynthesis anduptake, along with the levels of rate-limiting heme biosynthetic enzymesand heme transporters, are upregulated in NSCLC cells relative tonon-tumorigenic lung cells (FIG. S1A-B). This elevation causes theelevation of heme biosynthesis and uptake in NSCLC cells (FIGS. 1A-C).Increased heme biosynthesis and uptake in turn lead to elevatedmitochondrial heme levels (FIG. 1F). Based on the levels of hemesynthesis in normal medium and medium with heme depleted, the inventorestimates that NSCLC cells obtain about ⅔ of heme via de novo synthesisand about ⅓ via uptake from the medium (FIG. S8J). NSCLC cells are knownto require serum for growth in culture while normal lung epithelialcells grow better in the absence of serum (25, 48). Fetal bovine serumused to culture NSCLC cells, like human blood, contains approximately 20μM cell—free heme (49, 50). Thus, both in vitro in culture and in vivoin mice and humans, tumor cells have ample supply of heme from themedium or circulation. Heme degradation is also elevated in some NSCLCcells (FIG. 1D), and the inhibition of heme degradation by SnPP reducedcolony formation in NSCLC cells (see FIG. 4F & FIG. S7F). SnPP has beenshown to be a strong inhibitor for the activities of heme oxygenases(51). This result is consistent with other studies indicating a role ofheme degradation in promoting tumorigenesis (52, 53). For example, aprevious study showed that inhibition of heme degradation is lethal tohereditary leiomyomatosis and renal-cell cancer cells when fumaratehydratase is deficient (52). Very likely, elevated heme degradation incancer cells promotes tumorigenic functions by increasing the productionof potent antioxidants bilirubin and beliverdin, as well as iron.

Elevated mitochondrial heme levels can potentially influencemitochondrial OXPHOS in two ways: (1) by increasing the pool of hemewhich is incorporated into OXPHOS complexes and other hemoproteins, and(2) by upregulating the translocation and assembly of OXPHOS complexesand other enzymes. Therefore, the rates of oxygen consumption and ATPlevels are both elevated in NSCLC cells relative to non-tumorigeniccells (FIGS. 2A-B). Two proteins important for mitochondrial biogenesis,NRF2 and TFAM, are increased in NSCLC cells relative to non-tumorigeniccells (FIGS. 2C-D). This is consistent with a previous study showingthat loss of TFAM reduces tumorigenesis in an oncogenic Kras-drivenmouse model of lung cancer (54). Elevated heme biosynthesis and uptakeultimately lead to enhanced tumorigenic capabilities in NSCLC cells(FIGS. 4A-G & FIGS. S7A-I). Therefore, the data presented here and thosefrom previous studies all support the idea that heme is apro-tumorigenic metabolic and signaling molecule. Hemoproteins andenzymes that are required for OXPHOS are also pro-tumorigenic.Interestingly, a recent study from the authors' lab showed that viableNSCLC tumor cells resistant to the vascular disrupting agentcombretastatin A-4 phosphate exhibit further elevated levels ofhemoproteins and proteins and enzymes involved in heme metabolism (32).

Thus, the inventor expected that inhibitors of heme synthesis and uptakeshould suppress tumorigenesis and may overcome drug resistance. Indeed,the data presented here show that inhibition of heme synthesis bysuccinyl acetone (SA) or inhibition of heme uptake by HSPs reducestumorigenic functions of NSCLC cells (FIGS. 3A-C, FIGS. 4A-G, FIGS.S6A-D & FIGS. S7A-I). HSP2, which inhibits heme uptake more stronglythan HSP1, diminishes tumorigenic functions of NSCLC cells moststrongly. This raises the possibility that HSP2 can be a more effectiveagent against NSCLC cells than succinyl acetone. Indeed, HSP2 stronglysuppressed the growth of both orthotopically implanted NSCLC tumors andsubcutaneously implanted tumors (FIGS. 5A-H, FIGS. 6A-F & FIGS. S8A-J).Notably, addition of heme largely reverses the effects of SA and HSPs onproliferation, migration, invasion, and colony formation (FIG. 3C, FIGS.4A-G & FIGS. S7A-I). These results strongly support the idea that theeffects of SA and HSPs on NSCLC cell tumorigenic functions areattributable to their effects on heme synthesis and uptake,respectively.

The link between heme availability and NSCLC tumorigenesis is stronglysupported by data obtained from examining HSP2-treated tumors (FIGS.5A-H, FIGS. 6A-F & FIGS. S8A-J) and tumors formed by NSCLC cellsoverexpressing the rate-limiting heme synthesis enzyme ALAS1 or the hemeuptake protein/transporter SLC48A1 (FIGS. 7A-H). HSP2-treated tumors(FIGS. 5A-H, FIGS. 6A-F & FIGS. S8A-J) show lowered levels of OXPHOScomplex subunits (FIGS. 5E-5H), oxygen consumption (FIG. 6E), and ATPgeneration (FIG. 6F), indicating the effect of inhibited heme uptake onOXPHOS and ATP generation. Likewise, these data show that increasedlevels of ALAS1 or SLC48A1 cause increased heme synthesis (FIG. 7A) oruptake (FIG. 7B), respectively. This increase causes elevated oxygenconsumption (FIG. 7G) and ATP generation (FIG. 7H) in NSCLC tumors,which then promotes tumor growth, as shown by increased tumor sizes andmasses (FIGS. 7E-F).

Succinyl acetone has low toxicity to animals (55, 56). Likewise, datafrom the inventor's mouse studies suggest that HSP2 is not highly toxicto mice (FIGS. S8C-S8E). HSP2 did not affect red cell counts (FIG. S8C),hemoglobin levels (FIG. S8D), and ALT activity indicative of liverfunction (FIG. S8E). Moreover, HSP2 did not significantly affect theproliferation of HBEC30KT cell line representing normal lung epithelialcells in the concentration range that affected the proliferation ofNSCLC cells (FIG. 3C & FIG. S6A). Bacterial hemophore is notinternalized by host mammalian cells. Thus, it is expected that HSP2does not get into NSCLC cells, as indicated in FIGS. S5D-F. Notably, thedata clearly showed that HSP2 inhibits heme uptake and reducesmitochondrial heme levels in NSCLC cells (FIGS. 3A-C). The lack ofstrong blood toxicity of HSP2 is likely attributable to the lack of theneed for heme uptake in normal cells. For example, normal lung cells donot proliferate in the presence of serum, which containing cell-freeheme, whereas NSCLC cells need serum for proliferation andtumorigenicity (48). The data presented here show that the levels ofheme synthesis, uptake, oxygen consumption, and ATP are significantlylower in non-tumorigenic lung cells relative to NSCLC cells (FIGS. 1A-F& FIGS. 2A-D). Notably, during erythropoiesis, heme synthesis is inducedprior to and is essential for globin synthesis (34, 57). Erythroid hemesynthesis is very high and excessive. Previous experimental datasuggested that erythrocytes produce excess heme for export and transportto other organs (57, 58). Thus, heme uptake is not needed forerythropoiesis, and erythrocytes can provide heme for other cells andtissues, including tumors. Thus, it is not surprising that hemesequestration by HSP2 does not cause erythroid toxicity during thetreatment periods in mice (FIGS. 5A-H, FIGS. 6A-F & FIGS. S8A-J). It isalso worth noting that suppression of tumor growth should also lower thedemand for iron for tumor growth, thereby alleviating potential bloodtoxicity posed by HSP2.

Heme represents 97% of the functional iron pool in the human body. Ironcan contribute to both tumor initiation and progression (59). Indeed,the inventor's data show that iron chelator deferoxamine (DFX) inhibitedcolony formation in NSCLC cells (FIG. 4G & FIG. S7G). However, additionof iron does not reverse the effects of SA or HSPs on colony formation(FIGS. 4C-E & FIGS. S7C-E), because iron cannot reverse the effects ofSA and HSPs on heme synthesis or uptake. Heme and iron are linked: hemesynthesis requires iron, and heme degradation releases iron. However,likely due to their respective chemical properties, the main biologicalfunctions of heme iron and non-heme iron in living organisms may havebecome distinct. Due to its unique property for binding oxygen, theprimary functions of heme iron are for oxygen utilization, metabolism,and detoxification, particularly in OXPHOS for ATP generation. Non-hemeiron, however, often exists in proteins and enzymes as iron-sulfurcluster, and has essential functions in DNA replication, repair, andcell cycle (59, 60). Thus, both iron depletion and heme depletion canhave anti-tumor effects, but the mechanisms largely differ. Irondeficiency causes anemia, because red cells account for 80% offunctional iron needed in humans or mammals (34). In humans and mammals,the need of other cells for iron can presumably be met by taking a smallamount of iron from red cells. Indeed, iron availability affects hemesynthesis in erythroid cells, but not non-erythroid cells. Furthermore,heme synthesis in erythroid and non-erythroid cells involves differentALASs and regulatory modes (34, 57). Thus, lowering heme availabilityshould have more selective effects on NSCLC cells. Thus, targeting hemeuptake and/or heme synthesis may provide a new and effective strategyfor the treatment of NSCLC and perhaps other cancers resistant toexisting therapies.

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Example 2 Materials and Methods

Purification of HeSPs, Protein Binding to Heme-Agarose Beads,Spectroscopic Analyses, Size Exclusion Chromatography, ProteaseSensitivity Assay.

HeSPs were purified with the pET11a expression system. The pET11aexpression vector for Y. pestis HasA residues 1-193 (HasA_(yp)) waskindly provided by Dr. Mario Rivera (University of Kansas)³⁰. HeSP2contains Q32H Y75M double mutations, HeSP2H contains Q32H Y75H doublemutations, and HeSP2C contains Q32H Y75C double mutations. The mutationswere generated with the QuikChange II Site-Directed Mutagenesis Kit(Agilent Technologies). The double mutations were generated with theexpression vector for HasA_(yp). HeSP2del is HeSP2 with 125-133 aa(FDSGKSMTE (SEQ ID NO: 11)) residues delated; HeSP2ec contains 1-128 aaresidues of HeSP2 fused with 133-196 aa residues of Erwinia carotovoraHasA; HeSP2pc contains 1-136aa residues of HeSP2 fused with 139-196 aaresidues of Pectobacterium carotovorum HasA; and HeSP2pf contains1-101aa residues of HeSP2 fused with 104-194 aa residues ofPectobacterium fluorenscens HasA. DNA sequences encoding the deletionand hybrid proteins were synthesized (gBlocks, Integrated DNATechnologies Inc) and cloned in pET11a expression system. All DNA cloneswere confirmed by sequencing (Eurofins Genomics LLC). To purify HeSPsfrom E. coli, BL21(DE3) bearing the pET-11a expression plasmids weregrown to A_(0.8), and induced with 1 mM isopropylβ-D-1-thiogalactopyranoside (IPTG) for 5 hours at 30° C. Cells werecollected and lysed with a French Press. HeSPs were purified with aQ-Sepharose Fast Flow column (GE Healthcare), followed by size exclusionchromatography, as described³⁰. All purified proteins were analyzed onSDS-PAGE gels.

To detect protein binding to heme with heme agarose beads (Sigma), 500pmol purified proteins were incubated with 20 μl beads in 60 μl hemebinding buffer (20 mM Tris pH 8.0, 500 mM NaCl, and 1% TritonX-100) for1 hour at 4° C. After incubation, the beads were pelleted bycentrifugation, and the supernatant was collected. The beads were thenwashed twice with heme binding buffer. Subsequently, proteins bound tothe beads and in the supernatant were electrophoresed on SDSpolyacrylamide gels and visualized by Coomassie blue staining. Hemeabsorbance spectra were measured with a Varian Cary® 50 UV-VisSpectrophotometer. Samples contained 10 μM protein and 5 μM Heme.Protein and heme were prepared in 20 mM Tris, pH 8.0 and 500 mM NaCl;the imidazole stock was adjusted to pH 8.0 with HCl. Each sample wasincubated for 30 seconds after the addition of heme, prior to absorbancemeasurement. Size exclusion chromatography to separate heme andheme-protein complexes were carried out using 1 ml Sephadex-G50 (Sigma)columns, as described³¹. For chymotrypsin sensitivity assay, 20 μMpurified proteins were pre-incubated with or without heme (35 μg/ml) for10 min in 20 mM Tris, pH8.0, 500 mM NaCl. Then, chymotrypsin was addedto the proteins for 10 min. The reactions were stopped by adding SDSloading buffer, and samples were analyzed by SDS-PAGE.

Detection of Heme Transfer from Hemoglobin to HasA and HeSP2 UsingNative Polyacrylamide Gene Electrophoresis (PAGE).

Apo-HeSPs were mixed with hemoglobin to allow heme transfer.HeSP+hemoglobin were mixed and incubated for 30 minutes or indicatedtimes at 4° C. After incubation, buffer with 5% glycerol, 4 mM Tris (pH8.0), 40 mM NaCl, 4 mM MgCl2 was added, and samples were separated byelectrophoresis on native 5% polyacrylamide gels in⅓XTris-borate-ethylenediaminetetraacetic acid. Hemoglobin, apo-HeSP, andheme-bound HeSP were also analyzed in parallel for controls andcomparison. Native PAGE gels were subjected to heme staining andCoomassie blue staining. Heme staining was performed using the BioFX TMBOne Component HRP Microwell Substrate (Surmodics).

Cell Culture and Measurements of Heme Uptake, Cell Proliferation, andApoptosis.

NSCLC cell line H1299 (ATCC Cat #CRL-5803, RRID:CVCL_0060) was purchasedfrom ATCC, maintained in RPMI medium, and supplemented with 5%heat-inactivated, fetal bovine serum. H1299 expressing luciferase wasgenerated by infection with lentiviral particles bearing theEF1a-Luciferase (firefly) gene (AMSBIO)¹¹. Cell lines were authenticatedby Genetica and were found to be 96% identical to the standard(authentication requires >80%). Cell lines were tested for mycoplasmausing a MycoFluor™ Mycoplasma Detection Kit (Molecular Probes), and theresults were negative.

Cell proliferation was measured by counting live cells, as described¹¹.For measuring heme uptake, a fluorescent analog of heme, zincprotoporphyrin IX (ZnPP, Frontier Scientific Inc.), was used, asdescribed¹¹. Briefly, 10,000 NSCLC cells were seeded in 96-well plates.Cells were incubated for 3 hours with 60 μmol/L ZnPP in the presence orthe absence of 40 μmol/L HeSPs. Fluorescence intensity was measured witha Biotek Cytation 5 plate reader. Experiments were conducted intriplicates, and ZnPP uptake was normalized with total cellularproteins. Apoptosis was detected by using the ApoAlert™ Annexin V-FITCApoptosis Kit (Clontech). Cells were seeded in a 96-well black wallclear bottom plate at the density of 5,000 cells per well. After oneday, cells were treated with 20 μM HeSPs in the presence or the absenceof 20 μM heme in fresh medium for 6 days. Medium was changed every 3days. 6 days post treatment, apoptosis assay was performed according tomanufacturer's protocol. Fluorescent images were captured using BiotekCytation 5 plate reader.

Animals.

NOD/SCID mice (IMSR Cat #CRL:394, RRID:IMSR_CRL:394) were purchased fromCharles River Laboratories. Mice were bred and cared for in a Universityof Texas at Dallas specific pathogen-free animal facility in accordancewith NIH guidelines. All animal procedures were conducted underprotocols approved by Institutional Animal Care and Use Committee(IACUC) at the University of Texas at Dallas (UTD). Animals wereregularly examined for any signs of stress and euthanized according topreset criteria.

Treatment of Human NSCLC Xenografts in NOD/SCID Mice.

To generate subcutaneous models, 2×10⁶ H1299-Luc cells in serum-freemedium containing 50% Matrigel were injected subcutaneously into theleft flank region of 4-6 weeks old female NOD/SCID mice (n=6 per group).Mice were randomized into treatment groups that received saline (forcontrol) and various HeSPs (I.V. 25 mg/kg every 3 days), respectively.Body masses were recorded once every week. When the tumors reached 1 cm³(after ˜3 weeks of treatment), mice were euthanized by cervicaldislocation. Tumors were resected and weighed. Tumor tissues werebriefly homogenized and used for oxygen consumption rate and ATP assays.

For detecting the toxicity of HeSPs treatment on blood and liverfunctions, blood was obtained from mice via the sub-mandibular veinbefore sacrifice and was collected in blood collection tubes (BDmicrotainer tubes Cat #365963 and Cat #365974 from Fischer scientific).Serum samples were prepared and then used for determining hemoglobinlevels with a hemoglobin assay kit from Sigma-Aldrich (Cat #294MAK115-1KT) and ALT levels (alanine transaminase levels) with an ALTactivity assay kit from Sigma-Aldrich (MAK052-1KT), respectively. Wholeblood samples were used for counting red blood cells (RBCs) using ahemocytometer. No morphological differences were observed in red cellsfrom treated and untreated mice.

In vivo bioluminescence imaging (BLI).

Mice bearing lung tumor xenografts were imaged with an IVIS Lumina IIIIn Vivo Imaging system (Perkin Elmer). Briefly, mice were anesthetizedin the isoflurane chamber (2% isoflurane and oxygen), and luciferin(potassium salt; Perkin Elmer; 80 μl of 40 mg/ml) was administeredsubcutaneously between the scapulae. A BLI time course was acquired over30 mins (Exposure time: auto, F Stop: 1.2, Binning: medium). The imageswere quantified using Living Image software version 4.5.2 (PerkinElmer). Regions of interest (ROIs) were selected, and bioluminescencesignals integrated. The total bioluminescent signals (photon/sec) fromROIs of mice were calculated as specified by the manufacturer'sinstructions.

Measurement of Oxygen Consumption and ATP Levels.

Oxygen consumption was measured, as described previously¹¹. To measureoxygen consumption rates and ATP levels from freshly isolated tumors,subcutaneous tumors were surgically resected from mice and cut intosmall pieces. Tumors were weighed and homogenized immediately usingmechanical homogenizer to gain a homogenous cell suspension. Tissuedebris was removed by gentle centrifugation. Cells were suspended in 400ul of complete medium, and OCR was measured using a Clark-typeelectrode. ATP levels were measured with an ATP determination kit(Molecular probes). Liver cells were isolated and used to measure ATPlevels in the same manner. All experiments were carried out intriplicate, and the background luminescence was subtracted from themeasurement. ATP concentrations were calculated from the ATP standardcurve and normalized with the numbers of cells used. Both Oxygenconsumption rates and ATP levels were normalized with protein amounts.

Candida albicans Strain, Analyses of Pathogenic Functions, andMeasurement of Heme Uptake.

C. albicans strain SC5314 was kindly provided by Dr. Andrew Y Koh (UTSouthwestern Medical Center). C. albicans strain was grown overnight at37° C. in yeast extract-peptone-glycerol (YPG) medium. For themeasurement of heme uptake, a fluorescent analog of heme, zincprotoporphyrin IX (ZnPP, Frontier Scientific Inc.), was used. C.albicans strain was grown overnight at 37° C. in yeastextract-peptone-dextrose (YPD) medium, harvested by centrifugation,washed with PBS, and resuspended in PBS. Briefly, 10⁶ cells wereincubated for 1 hour with 60 μmol/L ZnPP in the presence or the absenceof 40 μmol/L HeSPs. Fluorescence intensity was measured with a BiotekCytation 5 plate reader. Experiments were conducted in triplicates, andZnPP uptake was normalized with total cell number.

For proliferation assays, the aforementioned overnight culture wasinoculated in yeast nitrogen base (YNB)-glycerol medium or YNB-glycerolcontaining indicated concentrations of HeSPs. C. albicans cellproliferation was determined by measuring optical density at 600 nm.Data presented are averages of four replicates for the readings obtainedat 96 hours. C. albicans biofilm formation was performed, as describedpreviously⁵⁰. Briefly, 100 μl of yeast suspension (10⁷ cells/ml) in PBSwas added in each well of a 96-well plate and the plate was incubated at37° C. for 90 min for adhesion. Each well was washed twice with PBS toremove non-adherent cells. 200 μl of synthetic complete medium orsynthetic complete medium containing HeSPs was added in each well andthe plate was incubated at 37° C. in an orbital shaker at 75 rpm for 48hours to form biofilm. Cells were then stained with 0.1% crystal violetfor 20 minutes, and washed three times with water followed byquantification. Quantification was performed by dissolving crystalviolet with 33% acetic acid. Absorbance was measured at 570 nm.

Results

Design and Biochemical Characterization of Heme-Sequestering Protein 2(HeSP2).

The hemophore HasA proteins are a family of highly conserved smallproteins without homology to other known proteins (FIG. 8a )²⁹. They arefound in several bacteria, such as Serratia marcescens, Yersinia pestis,and Pseudomonas fluorescens. An initial test with purified HasA proteinfrom Yersinia pestis showed that HasA_(yp) exhibits some, but notstatistically significant, anti-tumor activity (see FIGS. S9 a-b).Because HasA's native function is not simply to sequester heme, theinventor reasoned that altered HasA proteins that retain heme-bindingcapabilities but with altered structural conformation may have betteranti-tumor activity. She therefore made neutral changes in two keyresidues Q32 and Y75 that can directly contact heme, in the heme-bindingpocket³⁰ (see HeSP2 in FIG. 8b ). The Q32H change should not affect hemebecause H is the residue at that position in several HasA proteins (FIG.8a ). Additionally, the inventor made the Y75M change. Because both Yand M can chelate heme iron, this change should not affect heme binding.Indeed, she showed that HeSP2, like wild-type HasA_(yp), bind to hemestrongly using three previously established methods of detecting hemebinding³¹⁻³³. First, binding to heme agarose beads showed that HeSP2,like wild-type HasA, bound to heme strongly (FIG. 9a ). Second, HeSP2binding to heme shifted the peak of heme absorption Soret band from 385nm to 408 nm (FIG. 9b ). Third, size exclusion chromatography showedthat heme eluted together with HeSP2, while heme per se was not eluted(FIG. 9c ).

Next, the inventor examined the effects of the residue changes in HeSP2on protein conformation by detecting protease sensitivity. As expected,heme did not considerably affect the sensitivity of β-amylase, whichdoes not interact with heme (see FIG. 9a ), to chymotrypsin (FIG. 10a ).As shown in FIG. 10b , heme binding to wild-type HasA_(yp) made it moreresistant to chymotrypsin digestion, indicating altered conformation byheme binding. Interestingly, while the Q32H Y75M change in HeSP2appeared to make HeSP2 more resistant to chymotrypsin, heme binding didnot considerably alter the sensitivity of HeSP2 to chymotrypsin (FIG.10c ). Overall, the HeSP2-heme complex appeared to be more sensitive tochymotrypsin than the HasA_(yp)-heme complex (compare FIGS. 10b-c ).These results suggest that the HeSP2-heme complex has a differentconformation compared to the wild-type HasA-heme complex. Such a changein conformation may alter its anti-tumor property.

Further, the inventor directly showed that HeSP2 can effectively extractheme from hemoglobin. As shown in FIG. 10d , apo-HeSP2 and heme-boundHeSP2 migrated differently on a native gel (compare lanes 2 and 3 inFIG. 10d ). The presence of heme was confirmed by using heme stainingwith TMB (N,N,N′,N′-tetramethylbenzidine), as described³⁴. When HeSP2was mixed with hemoglobin, heme was transferred to HeSP2 (see lanes 4-7,FIG. 10d ). The transfer of heme was instantaneous (see FIG. S10 a).Further, HeSP2 appeared to be more effective at extracting heme fromhemoglobin than wild-type HasA (compare FIG. 9d with FIG. S10 b).

Generation of Additional HeSPs and their Effects on Lung Cancer Cells.

To generate more HeSPs that may have anti-tumor activity, the inventoralso changed Y75 to H and C, both of which can chelate heme, leading totwo more HeSPs, HeSP2H and HeSP2C (see FIG. 8b ). In addition, a loopregion (residues 125-133) in HasA_(yp) appears to be dispensable asshown in the structure of HasA_(yp) ³⁰. Thus, she deleted residues125-133 to generate HeSP2del (FIG. 8b ). Further, she generated threehybrid proteins with sequences from HasA proteins of Yersinia pestis,Pseudomonas fluorescens, Envinia carotovora, and Pectobacteriumcarotovorum. Notably, the latter three are not pathogenic to humans³⁵(see FIGS. 8a-b ). HasA sequences from such bacteria may offer differentpharmacological properties. The residues in the heme-binding pockets ofHasA proteins are highly conserved (FIG. 8a ). Thus, swapping andcombining various regions of different HasA proteins may allow us togenerate HeSPs with different immunogenic or pharmacological propertieswhile they all retain high heme-sequestration capability. Thus, theinventor selected residue positions with turns (without beta sheet oralpha helix) as fusion points and generated three hybrid proteins,HeSP2ec, HeSP2pc, and HeSP2pf (FIG. 8b ).

The inventor showed that all new HeSPs—HeSP2H, HeSP2C, HeSP2del,HeSP2ec, HeSP2pc, and HeSP2pf—bind to heme strongly, by examining hemeagarose binding (FIG. S11), heme absorption spectrum (FIG. S12), andsize exclusion chromatography (FIG. S13). All designed HeSPssignificantly inhibited heme uptake in NSCLC cells (FIG. 514a ).Importantly, all HeSPs inhibited NSCLC cell proliferation strongly, andaddition of heme largely reversed the effect of HeSPs (FIG. 11a ),indicating that the effects of HeSPs are attributable to hemesequestration. As expected, monitoring of HeSP2 incubated with NSCLCcells showed that HeSP2 remained stable in the medium even afterprolonged incubation (FIG. S14 b). The dose responses of NSCLC cellproliferation to HeSP treatments are shown in FIGS. 515a-g . Notably, asshown in FIG. 11b , treatment with HeSP2 caused apoptosis in NSCLCcells. Addition of heme reversed apoptosis, indicating that apoptosis isattributable to heme sequestration. Other HeSPs also caused apoptosis inNSCLC cells, and addition of heme reversed apoptosis (FIG. S16). Theseresults show that HeSPs can effectively inhibit heme uptake, suppresscancer cell proliferation, and induce apoptosis in NSCLC cells.

HeSPs Effectively Suppress NSCLC Tumor Growth in Mice and DiminishOxygen Consumption and ATP Generation in Tumors.

The inventor directly examined the effectiveness of HeSPs to suppresstumor growth in vivo. To this end, she used subcutaneously implantedhuman NSCLC xenografts in NOD/SCID mice (FIG. S13). Data in FIGS. 12a-bshow that all HeSPs significantly suppressed tumor growth, with theeffect of HeSP2 being the strongest. The treatments of HeSPs did notcause significant changes in the whole-body masses of mice (FIG. 12c ).The measurements of oxygen consumption rates (OCR) (FIG. 12d ) and ATPlevels (FIG. 12e ) in isolated tumor cells from mice showed that allHeSPs substantially reduced OCR and ATP generation in tumors.Importantly, treatment with HeSPs did not significantly affect red cellcounts (FIG. S17 a) and hemoglobin levels (FIG. S17 b) in the blood.Likewise, the treatments did not significantly affect liver functionshown by alanine transaminase (ALT) activity (FIG. S17 c). Thetreatments with HeSPs also did not significantly change ATP levels inliver cells isolated from mice (supplementary FIG. S17 d).

HeSPs Significantly Inhibit the Proliferation and Biofilm Formation inCandida albicans.

In humans, >95% of the functional iron is in the form of heme, most ofwhich is in hemoglobin molecules¹⁶. As such, pathogenic microbes havedeveloped robust machineries to obtain iron fromheme/hemoglobin^(5, 6, 29) . C. albicans uses a heme assimilation systeminvolving a conserved family of proteins, Rbt5, Rbt51/Pga10, Pga7, andCsa2^(7, 37, 38). These proteins are known to be important for C.albicans virulence^(7, 38, 39). Thus, the inventor tested whether HeSPscan influence C. albicans heme uptake, proliferation, and biofilmformation. As expected, HeSPs reduced heme uptake in C. albicanssignificantly (FIG. S18). As shown in FIG. 13a , HeSPs suppressed C.albicans proliferation significantly, and addition of heme largelyreversed the effects of HeSPs, indicating that the suppression isattributable to heme sequestration. Likewise, HeSPs inhibited C.albicans biofilm formation significantly (FIG. 13b ). Addition of hemeconsiderably reversed the effects of HeSPs on biofilm formation,indicating that heme sequestration contributed to the suppression ofbiofilm formation (FIG. 13b ). Together, these results suggest that hemesequestration can be a viable strategy to suppress the virulence of C.albicans.

Discussion

Heme has three major classes of functions in living organisms: (1) Hemeserves as an essential iron source and metallonutrient for organismsranging from pathogenic bacteria to humans^(5, 10, 28, 29). (2) Hemeserves as a prosthetic group in proteins and enzymes involved in oxygenmetabolism²⁰⁻²². (3) Heme serves as a signaling molecule regulatingdiverse molecule and cellular processes including transcription,translation, and microRNA processing^(14-17, 19). To carry out thesefunctions, heme needs to interact and bind, stably or transiently, todiverse cellular and extracellular proteins in living organisms. Thus,many studies have been carried out to characterize heme-proteininteractions and design peptides and proteins that bind to heme stablyor transiently^(17, 40, 41). In the past three decades, intense researchhas been carried out to design heme-binding proteins or maquettes,particularly those with four-helix bundle or β-sheet peptides⁴⁰⁻⁴³.However, these heme-binding proteins generally have heme-bindingaffinity in the nano to micro molar range^(40, 41), much lower than HasAproteins⁴. These designed proteins would not be able to extract andsequester heme from heme proteins such as hemoglobin. While humanhemopexin also binds to heme with pM affinity, heme-bound hemopexin canbe internalized by human cells and utilized as an iron source⁴⁴. Thus,hemopexin-like proteins would not be effective in sequestering heme fromhuman tumor cells.

Here, the inventor shows that HeSP2 is very effective at extracting hemefrom hemoglobin (FIG. 10d ). While previous experimental evidencesuggested that wild-type HasA can extract heme from hemoglobin⁴⁵, thedata shown in FIG. S10 b (compare to FIG. 10d ) suggest that wild-typeHasA is less effective than HeSP2 in extracting heme from hemoglobin.The increased capability of HeSP2 to extract heme is likely attributableto the difference in protease sensitivity (indicative of proteinconformation) induced by heme (FIGS. 10b-c ). While heme binding towild-type HasA_(yp) caused a strong increase in the resistance of theprotein to chymotrypsin (FIG. 10b ), heme binding to HeSP2 did notconsiderably change the sensitivity of the protein to chymotrypsin (FIG.10c ). This suggests that HeSP2 may have adopted a conformation that canreadily grip heme. The increased capability of HeSP2 to extract heme isalso consistent with its potent anti-tumor activity (FIG. 12a ) whilewild-type HasA did not exhibit statistically significant anti-tumoractivity (FIG. S1 a-b). Two other variants of HeSP2, HeSP2H and HeSP2C(FIG. 8b ), also exhibited significant anti-tumor activity and inhibitedoxygen consumption and ATP generation in tumor cells (FIGS. 12a-b ).Together, these results suggest that the heme binding pocket of HeSP2likely represents an optimized structure for heme extraction andsequestration.

HeSP2del containing an internal deletion of a loop (residues 124-133)outside the HasA main structure³⁰ (FIG. 8b ) was also stably expressedand exhibited strong activity in heme binding and inhibition of hemeuptake (FIGS. S10-13 a). HeSP2del exhibited significant anti-tumoractivity and inhibited oxygen consumption and ATP generation in tumorcells (FIGS. 12a-e ). However, shorter versions of HeSP2 with deletionof HasA residues at the C-terminus cannot be stably expressed, likelybecause these residues are part of the HasA compact structure, and theirdeletion destabilizes the overall structure. Nonetheless, these resultssuggest that HeSP2 variants with anti-tumor activity can be generated bymaking changes that do not disrupt the overall HasA protein structure.This idea is further tested by the generation of three hybrid HasAproteins: HeSP2pc, HeSP2ec, HeSP2pf (FIG. 8b ). They exhibited strongactivity in heme binding and inhibition of heme uptake (SupplementaryFIGS. 10-13 a), as well as significant anti-tumor activity (FIGS. 12a-b). These results show that functional HeSPs with strong anti-tumoractivity but potentially different pharmacological properties can begenerated by making hybrid HasA proteins.

It is worth noting that these engineered HeSPs can be applied for thetreatment of other diseases relating to heme. Here, the inventor testedthe effect of HeSPs on C. albicans because C. albicans uses heme as anutrient^(5, 6). She found that HeSPs inhibited heme uptake (FIG. S18),cell proliferation (FIG. 13a ), and biofilm formation (FIG. 13b ) in C.albicans. Importantly, excess heme have been shown to increase the risksof cardiovascular diseases and diabetes²⁸. Thus, this strategy of hemesequestration can potentially be applied to the treatment of thesecommon diseases.

Importantly, HeSPs did not significantly affect blood and liverfunctions. Treatment with HeSPs in mice did not cause significantchanges in RBC counts (FIG. S17 a), hemoglobin levels (FIG. S17 b), ALT(alanine aminotransferase) activity (FIG. S17 c), or liver cell ATPlevels (FIG. S17 d). These results are consistent with previous studiesshowing that the inhibitor of heme synthesis, succinyl acetone, has lowtoxicity to animals⁴⁶. Heme synthesis in erythroid and liver cells isvery high⁴⁷. Heme uptake is generally not needed for normal cells¹¹.Notably, previous studies indicated that erythrocytes can provide hemefor other cells and tissues^(8, 48). Thus, it is not surprising thatheme sequestration by HeSPs did not cause erythroid and liver toxicityduring the treatment periods in mice. Heme and iron are linked: hemesynthesis requires iron, and heme degradation releases iron. However,likely due to their respective chemical properties, the main biologicalfunctions of heme iron and non-heme iron in living organisms may havebecome distinct. Due to its unique property for binding oxygen, theprimary functions of heme iron are for oxygen utilization, metabolism,and detoxification, particularly in OXPHOS for ATP generation. Non-hemeiron, however, often exists in proteins and enzymes as iron-sulfurcluster, and has essential functions in DNA replication, repair, andcell cycle⁴⁹. Thus, both iron depletion and heme depletion can haveanti-tumor effects, but the mechanisms largely differ¹¹. Notably,suppression of tumor growth should also lower the demand for iron fortumor growth, thereby alleviating potential blood toxicity posed by HeSPtreatment. Taken together, these results show that the strategy of hemesequestration and the use of HeSPs can be applied in the prevention andtreatment of common diseases, including cancer and infection.

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The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe disclosure claimed. Thus, it should be understood that although thepresent disclosure has been specifically disclosed by preferredembodiments, exemplary embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this disclosure as defined by theappended claims. The specific embodiments provided herein are examplesof useful embodiments of the present disclosure and it will be apparentto one skilled in the art that the present disclosure may be carried outusing a large number of variations of the devices, device components,methods steps set forth in the present description. As will berecognized by one of skill in the art, methods and devices useful forthe present methods can include a large number of optional compositionand processing elements and steps.

What is claimed:
 1. A recombinant heme sequestering peptide (HeSP)comprising one or more of: (a) an amino acid substitution in a hemebinding pocket at Y75; and/or (b) a fusion of heme binding protein (HBP)sequences from distinct heme binding proteins, wherein the HeSP is anHasA peptide selected from the group consistent of Yersinia pestis HasA,Erwinia carotovora HasA, Pectobacterium carotvorum HasA and Pseudomonasfluorescens HasA.
 2. The HSeP of claim 1, wherein the HBP comprisesYersinia pestis HasA sequences.
 3. The HeSP of claim 1, wherein saidHeSP exhibits (a) and (b).
 4. The HeSP of claim 1, wherein said HeSPexhibits only (a).
 5. The HeSP of claim 1, wherein said HeSP exhibitsonly (b).
 6. The HeSP of claim 1, wherein said HeSP has the amino acidsequence of SEQ ID NOS:
 10. 7. The HeSP of claim 1, wherein said HeSP isbound to zinc protopoprhyrin.
 8. A method of sequestering heme from anenvironment comprising contact said environment with an HeSP of claim 1,2, 3, 6 or
 7. 9. The method of claim 8, wherein said environment is abiological sample.
 10. The method of claim 8, wherein said environmentis a cell culture.
 11. The method of claim 8, wherein said environmentis a surface.
 12. The method of claim 11, wherein the surface is aplate, tube, or well surface.
 13. The method of claim 8, wherein theenvironment is a surface of a medical device.
 14. The method of claim 8,wherein said HeSP is fixed to a support.
 15. The method of claim 14,wherein said support is a column matrix, a well, a plate, a slide, atube, a dipstick, a bead, or a nanoparticle.
 16. The method of claim 8,further comprising detecting sequestered heme.
 17. The method of claim16, further comprising quantifying the detected sequestered heme. 18.The method of claim 8, wherein the environment is an air handling deviceor system, a heating/cooling device or system, a water processing deviceor system, a water storage device or system, a water transport device orsystem, or a food processing device or system.
 19. A method of treatinga cancer that exhibits elevated oxidative phosphorylation in a subjectcomprising administering to said subject an HeSP of claim 1, 2, 3, 6 or7.
 20. The method of claim 19, wherein said cancer is lung cancer, coloncancer, head & neck cancer, brain cancer, pancreatic cancer, prostatecancer, ovarian cancer, testicular cancer, uterine cancer, breastcancer, skin cancer, lymphoma, or leukemia.
 21. The method of claim 20,wherein said lung cancer is non-small cell lung cancer.
 22. The methodof claim 20, wherein said breast cancer is triple negative breastcancer.
 23. The method of claim 20, wherein said skin cancer ismelanoma.
 24. The method of claim 19, wherein said cancer is a recurrentcancer, drug resistant cancer, primary cancer or metastatic cancer. 25.The method of claim 19, further comprising treating said subject withanother cancer therapy.
 26. The method of claim 25, wherein theadditional cancer therapy is chemotherapy, radiotherapy, toxin therapy,hormonal therapy, or surgery.
 27. The method of claim 19, wherein saidHeSP is administered local to a cancer site.
 28. The method of claim 19,wherein said HeSP is administered regional to a cancer site.
 29. Themethod of claim 19, wherein said HeSP is administered systemically. 30.A method of treating a fungal disease in a subject comprisingadministering to said subject an HeSP of claim 1, 2, 3, 6 or
 7. 31. Themethod of claim 30, further comprising treating said subject withanother anti-fungal therapy.
 32. The method of claim 30, wherein saidHeSP is administered local to a site of infection.
 33. The method ofclaim 30, wherein said HeSP is administered regional to a site ofinfection.
 34. The method of claim 30, wherein said HeSP is administeredsystemically.